Viral chemokine-antigen fusion proteins

ABSTRACT

The present invention relates to a vaccine for increasing the immunogenicity of a tumor antigen thus allowing treatment of cancer, as well as a vaccine that increases the immunogenicity of a viral antigen, thus allowing treatment of viral infection, including immunodeficiency virus (HIV) infection. In particular, the present invention provides a fusion protein comprising a viral chemokine fused to either a tumor antigen or viral antigen which is administered as either a protein or nucleic acid vaccine to elicit an immune response effective in treating cancer or effective in treating or preventing viral infection.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 10/380,927, filedMar. 17, 2003, which is the U.S. National Stage of InternationalApplication No. PCT/US2001/029075, filed Sep. 17, 2001, which waspublished in English under PCT Article 21(2), and which claims thebenefit of priority of U.S. Provisional Application No. 60/233,067,filed Sep. 15, 2000. All of the above-listed applications are hereinincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a vaccine for increasing theimmunogenicity of a tumor antigen thus allowing treatment of cancer, aswell as a vaccine that increases the immunogenicity of a viral antigen,thus allowing treatment of viral infection, including immunodeficiencyvirus (HIV) infection. In particular, the present invention provides afusion protein comprising a viral chemokine fused to either a tumorantigen or viral antigen which is administered as either a protein ornucleic acid vaccine to elicit an immune response effective in treatingcancer or effective in treating or preventing viral infection.

BACKGROUND OF THE INVENTION

Tumor cells are known to express tumor-specific antigens on the cellsurface. These antigens are believed to be poorly immunogenic, largelybecause they represent gene products of oncogenes or other cellulargenes which are normally present in the host and are therefore notclearly recognized as nonself. Although numerous investigators havetried to target immune responses against epitopes from various tumorspecific antigens, none have been successful in eliciting adequate tumorimmunity in vivo (71).

Humans are particularly vulnerable to cancer as a result of anineffective immunogenic response (72). In fact, the poor immunogenicityof relevant cancer antigens has proven to be the single greatestobstacle to successful immunotherapy with tumor vaccines (73). Over thepast 30 years, literally thousands of patients have been administeredtumor cell antigens as vaccine preparations, but the results of thesetrials have demonstrated that tumor cell immunization has failed toprovide a rational basis for the design or construction of effectivevaccines. Even where patients express tumor-specific antibodies orcytotoxic T-cells, this immune response does not correlate with asuppression of the associated disease. This failure of the immune systemto protect the host may be due to expression of tumor antigens that arepoorly immunogenic or to heterologous expression of specific antigens byvarious tumor cells. The appropriate presentation of tumor antigens inorder to elicit an immune response effective in inhibiting tumor growthremains a central issue in the development of an effective cancervaccine.

Chemokines are a group of usually small secreted proteins (7-15 kDa)induced by inflammatory stimuli and are involved in orchestrating theselective migration, diapedesis and activation of blood-born leukocytesthat mediate the inflammatory response (23,26). Chemokines mediate theirfunction through interaction with specific cell surface receptorproteins (23). At least four chemokine subfamilies have been identifiedas defined by a cysteine signature motif, termed CC, CXC, C and CX₃C,where C is a cysteine and X is any amino acid residue. Structuralstudies have revealed that at least both CXC and CC chemokines sharevery similar tertiary structure (monomer), but different quaternarystructure (dimer) (120-124). For the most part, conformationaldifferences are localized to sections of loop or the N-terminus.

There remains a great need for a method of presenting tumor antigens,which are known to be poorly immunogenic, “self” antigens to a subject'simmune system in a manner that elicits an immune response powerfulenough to inhibit the growth of tumor cells in the subject. Thisinvention overcomes the previous limitations and shortcomings in the artby providing a fusion protein comprising a viral chemokine and a tumorantigen which can produce an in vivo immune response, resulting in theinhibition of tumor cells. There is also a continuing need for a methodof presenting poorly antigenic viral antigens to a subject's immunesystem, particularly as relates to viral antigens such as HIV antigens.This invention also overcomes previous shortcomings in the field ofviral vaccine development by providing a fusion protein comprising aviral chemokine and a viral antigen which is effective as a vaccine fortreating or preventing viral infection.

SUMMARY OF THE INVENTION

The present invention provides a fusion polypeptide comprising a viralchemokine and a tumor antigen. In a preferred embodiment, the viralchemokine can be MIPI, MIPII, MIPIII, or any of the other viralchemokines set forth in Table 1, and the tumor antigen can be a B celltumor antigen or MUC-1.

The present invention also provides a fusion polypeptide comprising aviral chemokine and a viral antigen. In a preferred embodiment, theviral chemokine can be MIPI, MIPII, MIPIII, or any of the other viralchemokines set forth in Table 1, and the viral antigen can be an HIVantigen, such as gp120, gp160, gp41, an active fragment of gp120, anactive fragment of gp160 and/or an active fragment of gp41.

In addition, the present invention provides a method of producing animmune response in a subject, comprising administering to the subjectany of the fusion polypeptides of this invention, comprising a viralchemokine and viral antigen, or a viral chemokine and a tumor antigen,either as a protein or a nucleic acid encoding the fusion polypeptide.

Also provided is a method of treating a cancer in a subject comprisingadministering to the subject any of the fusion polypeptides of thisinvention, comprising a viral chemokine and a tumor antigen, either as aprotein or a nucleic acid encoding the fusion polypeptide.

The invention also provides a method of treating or preventing a viralinfection in a subject, comprising administering to the subject any ofthe fusion polypeptides of this invention, comprising a viral chemokineand a viral antigen, either as a protein or a nucleic acid encoding thefusion polypeptide.

Further provided is a method of treating or preventing HIV infection ina subject, comprising administering to the subject any of the fusionpolypeptides of this invention, comprising a viral chemokine and a humanimmunodeficiency virus (HIV) antigen, either as a protein or a nucleicacid encoding the fusion polypeptide.

A method of treating a B cell tumor in a subject is also provided,comprising administering to the subject any of the fusion polypeptidesof this invention, comprising a viral chemokine and a B cell tumorantigen, either as a protein or a nucleic acid encoding the fusionpolypeptide.

Various other objectives and advantages of the present invention willbecome apparent from the following detailed description.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used in the claims, “a” can include multiples. For example, “a cell”can mean a single cell or more than one cell. Also, unless otherwiseindicated, all references herein to a “chemokine” refer to a viralchemokine.

The present invention is based on the unexpected discovery that theadministration of a fusion protein comprising a viral chemokine and atumor antigen or administration of a nucleic acid encoding a fusionprotein comprising a viral chemokine and a tumor antigen yields aneffective and specific anti-tumor immune response by converting a “self”tumor antigen into a potent immunogen by binding to a viral chemokinemoiety. A further unexpected discovery of the present invention is thatthe viral chemokine-tumor antigen fusion polypeptide vaccine of thisinvention is superior to a human chemokine-tumor antigen fusionpolypeptide vaccine, as it provides a more potent immune response anddoes not cause the production of auto-antibodies directed against thehuman chemokine moiety of the latter fusion polypeptide.

Thus, the present invention provides a fusion polypeptide comprising aviral chemokine and a tumor antigen. The fusion polypeptide can bepresent in a purified form and can induce an immune response against thetumor antigen and inhibit the growth of tumor cells expressing the tumorantigen. “Purified” as used herein means the polypeptide is sufficientlyfree of contaminants or cell components with which proteins normallyoccur to allow the peptide to be used therapeutically. It is notcontemplated that “purified” necessitates having a preparation that istechnically totally pure (homogeneous), but purified as used hereinmeans the fusion polypeptide is sufficiently pure to provide thepolypeptide in a state where it can be used therapeutically. As usedherein, “fusion polypeptide” means a polypeptide made up of two or moreamino acid sequences representing peptides or polypeptides fromdifferent sources. Also as used herein, “epitope” refers to a specificamino acid sequence of limited length which, when present in the properconformation, provides a reactive site for an antibody or T cellreceptor. The identification of epitopes on antigens can be carried outby immunology protocols that are standard in the art (74). As furtherused herein, “tumor antigen” describes a polypeptide expressed on thecell surface of specific tumor cells and which can serve to identify thetype of tumor. An epitope of the tumor antigen can be any site on theantigen that is reactive with an antibody or T cell receptor.

As used herein, “viral chemokine” means a protein encoded by a viruswhich orchestrates a chemotactic response typically after binding tospecific G-protein-coupled cell surface receptors on target cells (e.g.,antigen presenting cells (APC), such as dendritic cells, monocytes,macrophages, keratinocytes and B cells), comprising the selectivemigration, diapedesis and activation of leukocytes which mediate theinflammatory response.

Numerous viral chemokines have been identified, including, for example,chemokines produced by Kaposi's sarcoma-associated herpes virus(KSHV/HHV8), which are analogues of human chemokine MIP-1s. These aredesignated vMIPI, vMIPII and vMIPII. (128, 129). vMIPI is an agonistchemokine which binds to CCR8-expressing cells, e.g., T helper 2(T_(H)2) cells, while vMIPII is considered to be a broad spectrumantagonist of chemotaxis, and binds to several chemokine receptors suchas CCR1, CCR2, CCR5, CXCR4, and cytomegalovirus chemokine receptor US28. vMIPII can induce chemotaxis via binding to CCR3 and CCR8. vMIPIIIis an agonist which binds CCR4 expressed by T_(H)2-type T cells.

The viral chemokine of this invention can include, but is not limitedto, vMIPI, vMIPII, vMIPIII, and any of the viral chemokines set forth inTable 1 as well as any other viral chemokine now known or lateridentified (128). Additional examples of viral chemokines which may beused in the compositions and methods of the present invention are setforth in Lalani et al. (Immunology Today 21:100-106 (2000)), thecontents of which are hereby incorporated by reference in theirentirety.

It will be appreciated by one of skill in the art that viral chemokinescan include active fragments of viral chemokines which retain theactivity, including chemotaxis and inhibition of chemotaxis, of theintact molecule.

A viral chemokine consists of two structural portions: the aminoterminal portion and the carboxy terminal portion. The amino terminalportion is responsible for viral chemokine receptor binding and thecarboxy terminal end binds to heparin and heparin sulfate, for example,in the extracellular matrix and on the surface of endothelial cells. Theviral chemokine gene can be fragmented as desired and the fragments canbe fused to a specific marker gene encoding an antigen (e.g., Muc-1 VNT,lymphoma scFv, etc.). The fusion polypeptide comprising the viralchemokine fragment and the tumor or viral antigen can be produced andpurified as described herein and tested for immunogenicity according tothe methods provided herein. By producing several fusion polypeptideshaving viral chemokine fragments of varying size, the minimal sizechemokine fragment which imparts an immunological effect can beidentified.

The tumor antigen moiety of the fusion polypeptide of this invention canbe any tumor antigen now known or later identified as a tumor antigen.The appropriate tumor antigen used in the fusion polypeptide naturallydepends on the tumor type being treated. For example, the tumor antigencan be, but is not limited to human epithelial cell mucin (Muc-1; a 20amino acid core repeat for Muc-1 glycoprotein, present on breast cancercells and pancreatic cancer cells), the Ha-ras oncogene product, p53,carcino-embryonic antigen (CEA), the raf oncogene product, GD2, GD3,GM2, TF, sTn, MAGE-1, MAGE-3, tyrosinase, gp75, Melan-A/Mart-1, gp100,HER2/neu, EBV-LMP 1 & 2, HPV-F4, 6, 7, prostatic serum antigen (PSA),alpha-fetoprotein (AFP), CO17-1A, GA733, gp72, p53, the ras oncogeneproduct, proteinase 3, Wilm's tumor antigen-1, telomerase, HPV E7 andmelanoma gangliosides, as well as any other tumor antigens now known oridentified in the future. Tumor antigens can be obtained following knownprocedures or are commercially available (79). The effectiveness of thefusion protein in eliciting an immune response against a particulartumor antigen can be determined according to methods standard in the artfor determining the efficacy of vaccines and according to the methodsset forth in the Examples.

Additionally, the tumor antigen of the present invention can be anantibody which can be produced by a B cell tumor (e.g., B cell lymphoma;B cell leukemia; myeloma) or the tumor antigen can be a fragment of suchan antibody, which contains an epitope of the idiotype of the antibody.The epitope fragment can comprise as few as nine amino acids. Forexample, the tumor antigen of this invention can be a malignant B cellantigen receptor, a malignant B cell immunoglobulin idiotype, a variableregion of an immunoglobulin, a hypervariable region or complementaritydetermining region (CDR) of a variable region of an immunoglobulin, amalignant T cell receptor (TCR), a variable region of a TCR and/or ahypervariable region of a TCR.

In a preferred embodiment, the tumor antigen of this invention can be asingle chain antibody (scFv), comprising linked V_(H) and V_(L) domainsand which retains the conformation and specific binding activity of thenative idiotype of the antibody (27). Such single chain antibodies arewell known in the art and can be produced by standard methods and asdescribed in the Examples herein.

In addition, the tumor antigen of the present invention can be anepitope of the idiotype of a T cell receptor, which can be produced by aT cell tumor (e.g., T cell lymphoma; T cell leukemia; myeloma). Theepitope can comprise as few as nine amino acids.

As will be appreciated by those skilled in the art, the invention alsoincludes peptides and polypeptides having slight variations in aminoacid sequences or other properties. Such variations may arise naturallyas allelic variations (e.g., due to genetic polymorphism) or may beproduced by human intervention (e.g., by mutagenesis of cloned DNAsequences), such as induced point, deletion, insertion and substitutionmutantions. Minor changes in amino acid sequence are generallypreferred, such as conservative amino acid replacements, small internaldeletions or insertions, and additions or deletions at the ends of themolecules. Substitutions may be designed based on, for example, themodel of Dayhoff et al. (80). These modifications can result in changesin the amino acid sequence, provide silent mutations, modify arestriction site, or provide other specific mutations.

The fusion polypeptides can comprise one or more selected epitopes onthe same tumor antigen, one or more selected epitopes on different tumorantigens, as well as repeats of the same epitope, either in tandem orinterspersed along the amino acid sequence of the fusion polypeptide.The tumor antigen can be positioned in the fusion polypeptide at thecarboxy terminus of the viral chemokine, the amino terminus of viralchemokine and/or at one or more internal sites within the viralchemokine amino acid sequence. Additionally, the fusion polypeptide cancomprise more than one viral chemokine in any combination and in anyorder with the various tumor antigens described above.

It would be routine for an artisan to produce a fusion proteincomprising any viral chemokine region and any human tumor antigen (e.g.,human single chain antibody) region according to the methods describedherein, on the basis of the availability in the art of the nucleic acidand/or amino acid sequence of the viral chemokine of interest and thehuman tumor antigen of interest.

The present invention further provides a fusion polypeptide comprising afirst region comprising a viral chemokine selected from the groupconsisting of vMIPI, vMIPII and vMIPIII, and a second region comprisinga tumor antigen selected from the group consisting of human epithelialcell mucin (Muc-1), the Ha-ras oncogene product, p53, carcino-embryonicantigen (CEA), the raf oncogene product, GD2, GD3, GM2, TF, sTn, MAGE-1,MAGE-3, tyrosinase, gp75, Melan-A/Mart-1, gp100, HER2/neu, EBV-LMP 1 &2, HPV-F4, 6, 7, prostatic serum antigen (PSA), alpha-fetoprotein (AFP),C017-1A, GA733, gp72, p53, the ras oncogene product, HPV E7, melanomagangliosides, an antibody produced by a B cell tumor (e.g., B celllymphoma; B cell leukemia; myeloma), a fragment of such an antibody,which contains an epitope of the idiotype of the antibody, a malignant Bcell antigen receptor, a malignant B cell immunoglobulin idiotype, avariable region of an immunoglobulin, a hypervariable region or CDR of avariable region of an immunoglobulin, a malignant T cell receptor (TCR),a variable region of a TCR and/or a hypervariable region of a TCR.

For example, the present invention provides a fusion polypeptidecomprising an scFv cloned from a human subject's biopsy tumor materialor from a hybridoma cell line producing a lymphoma antibody and a viralchemokine moiety (e.g., vMIPI, vMIPII, vMIPIII, etc.). In addition, thepresent invention provides a viral chemokine fused with the Muc-1 coreepitope of human breast cancer or human pancreatic cancer. Muc-1 is aglycoprotein (Mr>200,000) abundantly expressed on breast cancer cellsand pancreatic tumor cells. A variable number of tandem (VNT) repeats ofa 20 amino acid peptide (PDTRPAPGSTAPPAHGVTSA; SEQ ID NO:1) include Band T cell epitopes. Thus, the present invention provides a fusionprotein comprising vMIPI and Muc-1 VNT and vMIPII and Muc-1 VNT. Theexpression vector is designed so that a VNT can be changed by routinecloning methods to produce a fusion polypeptide comprising vMIPI orvMIPII fused with a Muc-1 VNT dimer, trimer, tetramer, pentamer,hexamer, etc.

In specific embodiments, the present invention also provides a fusionpolypeptide comprising vMIPI and human Muc-1, a fusion polypeptidecomprising vMIPII and human Muc-1, and a fusion polypeptide comprisingvMIPIII and human Muc-1 and human Muc-1.

The present invention further provides a fusion polypeptide comprising aviral chemokine (e.g., vMIPI, vMIPII, vMIPIII, etc.) and a scFv whichrecognizes tumor antigens, such as idiotype-specific scFv, Muc-1, etc.Such a fusion polypeptide would allow migration, recruitment andactivation of specialized cells of the immune system, such as naturalkiller (NK) cells, macrophages, dendritic cells (DC), polymorphonuclear(PMN) leukocytes, cytotoxic lymphocytes (CTL), etc., which would destroythe target cell.

The fusion polypeptide of this invention can further comprise a spacersequence between the viral chemokine and the tumor antigen or viralantigen, which can have the amino acid sequence EFNDAQAPKSLE (SEQ IDNO:2), or an amino acid sequence with conservative substitutions suchthat it has the same functional activity as the amino acid sequence ofSEQ ID NO:2, which allows for retention of the correct folding of thetumor antigen region of the polypeptide.

In addition, the present invention provides a composition comprising thefusion polypeptide of this invention and a suitable adjuvant. Such acomposition can be in a pharmaceutically acceptable carrier, asdescribed herein. As used herein, “suitable adjuvant” describes asubstance capable of being combined with the fusion polypeptide toenhance an immune response in a subject without deleterious effect onthe subject. A suitable adjuvant can be, but is not limited to, forexample, an immunostimulatory cytokine, SYNTEX adjuvant formulation 1(SAF-1) composed of 5 percent (wt/vol) squalene (DASF, Parsippany,N.J.), 2.5 percent Pluronic, L121 polymer (Aldrich Chemical, Milwaukee),and 0.2 percent polysorbate (Tween 80, Sigma) in phosphate-bufferedsaline. Other suitable adjuvants are well known in the art and includeQS-21, Freund's adjuvant (complete and incomplete), alum, aluminumphosphate, aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE) and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trealosedimycolate and cell wall skeleton (MPL+TDM+CWS) in 2% squalene/Tween 80emulsion.

The adjuvant, such as an immunostimulatory cytokine can be administeredbefore the administration of the fusion protein or nucleic acid encodingthe fusion protein, concurrent with the administration of the fusionprotein or nucleic acid or up to five days after the administration ofthe fusion polypeptide or nucleic acid to a subject. QS-21, similarly toalum, complete Freund's adjuvant, SAF, etc., can be administered withinhours of administration of the fusion protein or nucleic acid.

Furthermore, combinations of adjuvants, such as immunostimulatorycytokines can be co-administered to the subject before, after orconcurrent with the administration of the fusion polypeptide or nucleicacid. For example, combinations of adjuvants, such as immunostimulatorycytokines, can consist of two or more of immunostimulatory cytokines ofthis invention, such as GM/CSF, interleukin-2, interleukin-12,interferon-gamma, interleukin-4, tumor necrosis factor-alpha,interleukin-1, hematopoietic factor flt3L, CD40L, B7.1 co-stimulatorymolecules and B7.2 co-stimulatory molecules. The effectiveness of anadjuvant or combination of adjuvants may be determined by measuring theimmune response directed against the fusion polypeptide with and withoutthe adjuvant or combination of adjuvants, using standard procedures, asdescribed herein.

Furthermore, the present invention provides a composition comprising thefusion polypeptide of this invention or a nucleic acid encoding thefusion polypeptide of this invention and an adjuvant, such as animmunostimulatory cytokine or a nucleic acid encoding an adjuvant, suchas an immunostimulatory cytokine. Such a composition can be in apharmaceutically acceptable carrier, as described herein. Theimmuno-stimulatory cytokine used in this invention can be, but is notlimited to, GM/CSF, interleukin-2, interleukin-12, interferon-gamma,interleukin-4, tumor necrosis factor-alpha, interleukin-1, hematopoieticfactor flt3L, CD40L, B7.1 con-stimulatory molecules and B7.2co-stimulatory molecules.

The present invention further contemplates a fusion polypeptidecomprising a viral chemokine, or active fragment thereof, as describedherein and a viral antigen, which can be, for example, an antigen ofhuman immunodeficiency virus (HIV). An HIV antigen of this invention canbe, but is not limited to, the envelope glycoprotein gp120, the thirdhypervariable region of the envelope glycoprotein, gp120 of HIV-1 (thedisulfate loop V3), having the amino acid sequence:NCTRPNNNTRICRIRIQRGPGRA FVTIGKIGNMRQAHCNIS (SEQ ID NO:3), any otherantigenic fragment of gp120, the envelope glycoprotein gp160, anantigenic fragment of gp160, the envelope glycoprotein gp41 and/or anantigenic fragment of gp41. For example, the nucleic acid encoding theV3 loop can be fused to the 3′ end of the nucleic acid encoding a viralchemokine (e.g., vMIPI, vMIPII, or vMIPIII) directly or separated bynucleic acid encoding a spacer sequence. The viral chemokine-V3 loopfusion polypeptide can be produced in an expression system as describedherein and purified as also described herein.

In specific embodiments, the present invention provides a fusionpolypeptide comprising a viral chemokine and a human immunodeficiencyvirus (HIV) antigen, wherein the viral chemokine can be vMIPI, vMIPII,vMIPIII, or any other viral chemokine, and wherein the HIV antigen canbe gp120, gp160, gp41, an active (i.e., antigenic) fragment of gp120, anactive (i.e., antigenic) fragment of gp160 and an active (i.e.,antigenic) fragment of gp41.

Further provided in this invention is fusion polypeptide comprisingvMIPI and HIV gp120, a fusion polypeptide comprising vMIPII and HIVgp120, and a fusion polypeptide comprising vMIPIII and HIV gp120.

An isolated nucleic acid encoding the fusion polypeptides of thisinvention as described above is also provided. By “isolated nucleicacid” is meant a nucleic acid molecule that is substantially free of theother nucleic acids and other components commonly found in associationwith nucleic acid in a cellular environment. Separation techniques forisolating nucleic acids from cells are well known in the art and includephenol extraction followed by ethanol precipitation and rapidsolubilization of cells by organic solvent or detergents (81).

The nucleic acid encoding the fusion polypeptide can be any nucleic acidthat functionally encodes the fusion polypeptide. To functionally encodethe polypeptide (i.e., allow the nucleic acid to be expressed), thenucleic acid can include, for example, expression control sequences,such as an origin of replication, a promoter, an enhancer and necessaryinformation processing sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites and transcriptional terminator sequences.Preferred expression control sequences are promoters derived frommetallothionine genes, actin genes, immunoglobulin genes, CMV, SV40,adenovirus, bovine papilloma virus, etc. A nucleic acid encoding aselected fusion polypeptide can readily be determined based upon thegenetic code for the amino acid sequence of the selected fusionpolypeptide and many nucleic acids will encode any selected fusionpolypeptide. Modifications in the nucleic acid sequence encoding thefusion polypeptide are also contemplated. Modifications that can beuseful are modifications to the sequences controlling expression of thefusion polypeptide to make production of the fusion polypeptideinducible or repressible as controlled by the appropriate inducer orrepressor. Such means are standard in the art (81). The nucleic acidscan be generated by means standard in the art, such as by recombinantnucleic acid techniques, as exemplified in the examples herein and bysynthetic nucleic acid synthesis or in vitro enzymatic synthesis.

A vector comprising any of the nucleic acids of the present inventionand a cell comprising any of the vectors of the present invention arealso provided. The vectors of the invention can be in a host (e.g., cellline or transgenic animal) that can express the fusion polypeptidecontemplated by the present invention.

There are numerous E. coli (Escherichia coli) expression vectors knownto one of ordinary skill in the art useful for the expression of nucleicacid encoding proteins such as fusion proteins. Other microbial hostssuitable for use include bacilli, such as Bacillus subtilis, and otherenterobacteria, such as Salmonella, Serratia, as well as variousPseudomonas species. These prokaryotic hosts can support expressionvectors which will typically contain expression control sequencescompatible with the host cell (e.g., an origin of replication). Inaddition, any number of a variety of well-known promoters will bepresent, such as the lactose promoter system, a tryptophan (Trp)promoter system, a beta-lactamase promoter system, or a promoter systemfrom phage lambda. The promoters will typically control expression,optionally with an operator sequence and have ribosome binding sitesequences for example, for initiating and completing transcription andtranslation. If necessary, an amino terminal methionine can be providedby insertion of a Met codon 5′ and in-frame with the protein. Also, thecarboxy-terminal extension of the protein can be removed using standardoligonucleotide mutagenesis procedures.

Additionally, yeast expression can be used. There are several advantagesto yeast expression systems. First, evidence exists that proteinsproduced in a yeast secretion system exhibit correct disulfide pairing.Second, post-translational glycosylation is efficiently carried out byyeast secretory systems. The Saccharomyces cerevisiaepre-pro-alpha-factor leader region (encoded by the MFα-1 gene) isroutinely used to direct protein secretion from yeast (82). The leaderregion of pre-pro-alpha-factor contains a signal peptide and apro-segment which includes a recognition sequence for a yeast proteaseencoded by the KEX2 gene. This enzyme cleaves the precursor protein onthe carboxyl side of a Lys-Arg dipeptide cleavage-signal sequence. Thepolypeptide coding sequence can be fused in-frame to thepre-pro-alpha-factor leader region. This construct is then put under thecontrol of a strong transcription promoter, such as the alcoholdehydrogenase I promoter or a glycolytic promoter. The protein codingsequence is followed by a translation termination codon which isfollowed by transcription termination signals. Alternatively, thepolypeptide coding sequence of interest can be fused to a second proteincoding sequence, such as Sj26 or β-galactosidase, used to facilitatepurification of the fusion protein by affinity chromatography. Theinsertion of protease cleavage sites to separate the components of thefusion protein is applicable to constructs used for expression in yeast.

Efficient post-translational glycosylation and expression of recombinantproteins can also be achieved in Baculovirus systems in insect cells.

Mammalian cells permit the expression of proteins in an environment thatfavors important post-translational modifications such as folding andcysteine pairing, addition of complex carbohydrate structures andsecretion of active protein. Vectors useful for the expression ofproteins in mammalian cells are characterized by insertion of theprotein coding sequence between a strong viral promoter and apolyadenylation signal. The vectors can contain genes conferring eithergentamicin or methotrexate resistance for use as selectable markers. Theantigen and immunoreactive fragment coding sequence can be introducedinto a Chinese hamster ovary (CHO) cell line using a methotrexateresistance-encoding vector. Presence of the vector RNA in transformedcells can be confirmed by Northern blot analysis and production of acDNA or opposite strand RNA corresponding to the protein coding sequencecan be confirmed by Southern and Northern blot analysis, respectively. Anumber of other suitable host cell lines capable of secreting intactproteins have been developed in the art and include the CHO cell lines,HeLa cells, myeloma cell lines, Jurkat cells and the like. Expressionvectors for these cells can include expression control sequences, asdescribed above.

The vectors containing the nucleic acid sequences of interest can betransferred into the host cell by well-known methods, which varydepending on the type of cell host. For example, calcium chloridetransfection is commonly utilized for prokaryotic cells, whereas calciumphosphate treatment, lipofection or electroporation may be used forother cell hosts.

Alternative vectors for the expression of protein in mammalian cells,similar to those developed for the expression of human gamma-interferon,tissue plasminogen activator, clotting Factor VIII, hepatitis B virussurface antigen, protease Nexinl, and eosinophil major basic protein,can be employed. Further, the vector can include CMV promoter sequencesand a polyadenylation signal available for expression of insertednucleic acid in mammalian cells (such as COS7).

The nucleic acid sequences can be expressed in hosts after the sequenceshave been positioned to ensure the functioning of an expression controlsequence. These expression vectors are typically replicable in the hostorganisms either as episomes or as an integral part of the hostchromosomal DNA. Commonly, expression vectors can contain selectionmarkers, e.g., tetracycline resistance or hygromycin resistance, topermit detection and/or selection of those cells transformed with thedesired nucleic acid sequences (83).

Additionally, the fusion polypeptides and/or nucleic acids of thepresent invention can be used in in vitro diagnostic assays, as well asin screening assays for identifying unknown tumor antigen epitopes andfine mapping of tumor antigen epitopes.

Also provided is a method for producing a fusion polypeptide comprisinga viral chemokine, or an active fragment thereof and a tumor antigen orviral antigen, comprising cloning into an expression vector a first DNAfragment encoding a viral chemokine or active fragment thereof and asecond DNA fragment encoding a tumor antigen or viral antigen; andexpressing the DNA of the expression vector in an expression systemunder conditions whereby the fusion polypeptide is produced. Theexpression vector and expression system can be of any of the types asdescribed herein. The cloning of the first and second DNA segments intothe expression vector and expression of the DNA under conditions whichallow for the production of the fusion protein of this invention can becarried out as described in the Examples section included herein. Themethod of this invention can further comprise the step of isolating andpurifying the fusion polypeptide, according to methods well known in theart and as described herein.

Any of the fusion polypeptides, the nucleic acids and the vectors of thepresent invention can be in a pharmaceutically acceptable carrier and inaddition, can include other medicinal agents, pharmaceutical agents,carriers, diluents, adjuvants (e.g., immunostimulatory cytokines), etc.By “pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the selected antigen withoutcausing substantial deleterious biological effects or interacting in adeleterious manner with any of the other components of the compositionin which it is contained. Actual methods of preparing such dosage formsare known, or will be apparent, to those skilled in this art (84).

Thus, the present invention further provides a method for inducing animmune response in a subject capable of induction of an immune responseand preferably human, comprising administering to the subject an immuneresponse-inducing amount of the fusion polypeptide of this invention. Asused herein, “an immune response-inducing amount” is that amount offusion polypeptide which is capable of producing in a subject a humoraland/or cellular immune response capable of being detected by standardmethods of measurement, such as, for example, as described herein. Forexample, the antigenic polypeptide region can induce an antibodyresponse. The antibodies can treat or prevent a pathological or harmfulcondition in the subject in which the antibodies are produced or theantibodies can be removed from the subject and administered to anothersubject to treat or prevent a pathological or harmful condition. Thefusion polypeptide can also induce an effector T cell (cellular) immuneresponse which is effective in treating or preventing a pathological orharmful conditions in the subject.

In an embodiment wherein the antigen moiety of the fusion polypeptidecomprises an immunoglobulin light or heavy chain or a single chainantibody, the immune response can be the production in the subject ofanti-idiotype antibodies, which represent the image of the originalantigen and can function in a vaccine preparation to induce an immuneresponse to a pathogenic antigen, thereby avoiding immunization with theantigen itself (85). The anti-idiotype antibodies can treat or prevent apathological or harmful condition in the subject in which theanti-idiotype antibodies are produced or the anti-idiotype antibodiescan be removed from the subject and administered to another subject totreat or prevent a pathological or harmful condition.

Further provided is a method for inhibiting the growth of tumor cells ina subject, comprising administering to the subject a tumor cellgrowth-inhibiting amount of the fusion polypeptide of this invention.The subject of this method can be any subject in which a humoral and/orcellular immune response to a tumor can be induced, which is preferablyan animal and most preferably a human. As used herein, “inhibiting thegrowth of tumor cells” means that, following administration of thefusion polypeptide, a measurable humoral and/or cellular immune responseagainst the tumor cell epitope is elicited in the subject, resulting inthe inhibition of growth of tumor cells present in the subject. Thehumoral immune response can be measured by detection, in the serum ofthe subject, of antibodies reactive with the epitope of the tumorantigen present on the fusion polypeptide, according to protocolsstandard in the art, such as enzyme linked immunosorbent immunoassay(ELISA) and Western blotting protocols. The cellular immune response canbe measured by, for example, footpad swelling in laboratory animals,peripheral blood lymphocyte (PBL) proliferation assays and PBLcytotoxicity assays, as would be known to one of ordinary skill in theart of immunology and particularly as set forth in the availablehandbooks and texts of immunology protocols (86).

The present invention also provides a method of treating cancer in asubject diagnosed with cancer, comprising administering to the subjectan effective amount of the fusion polypeptide of the present invention.The cancer can be, but is not limited to B cell lymphoma, T celllymphoma, myeloma, leukemia, breast cancer, pancreatic cancer, coloncancer, lung cancer, renal cancer, liver cancer, prostate cancer,melanoma and cervical cancer.

Further provided is a method of treating a B cell tumor in a subjectdiagnosed with a B cell tumor, comprising administering an effectiveamount of the fusion polypeptide of this invention, which comprises anantibody or a fragment thereof, as described herein, in apharmaceutically acceptable carrier, to the subject.

In specific embodiments, the present invention also provides a method ofproducing an immune response in a subject, comprising administering tothe subject a composition comprising a fusion polypeptide of thisinvention and a pharmaceutically acceptable carrier and wherein thefusion polypeptide can be a fusion polypeptide comprising vMIPI andhuman Muc-1, a fusion polypeptide comprising vMIPII and human Muc-1 or afusion protein comprising vMIPII and human Muc-1.

Also provided is a method of producing an immune response in a subject,comprising administering to the subject a composition comprising anucleic acid encoding a fusion polypeptide of this invention and apharmaceutically acceptable carrier and wherein the fusion polypeptideis a fusion polypeptide comprising comprising vMIPI and human Muc-1, afusion polypeptide comprising vMIPII and human Muc-1, or a fusionprotein comprising vMIPIII and human Muc-1, under conditions whereby thenucleic acid of the composition can be expressed, thereby producing animmune response in the subject.

In further embodiments, the present invention also provides a method ofproducing an immune response in a subject, comprising administering tothe subject a composition comprising a fusion polypeptide of thisinvention and a pharmaceutically acceptable carrier and wherein thefusion polypeptide can be a fusion polypeptide comprising vMIPI and HIVgp120, or a fusion polypeptide comprising vMIPII and HIV gp120, or afusion polypeptide comprising vMIPIII and HIV gp120, thereby producingan immune response in the subject.

Also provided is a method of producing an immune response in a subject,comprising administering to the subject a composition comprising anucleic acid encoding a fusion polypeptide of this invention and apharmaceutically acceptable carrier and wherein the fusion polypeptideis a fusion polypeptide comprising vMIPI and HIV gp120, a fusionpolypeptide comprising vMIPII and HIV gp120, or a fusion polypeptidecomprising vMIPIII and HIV gp120 under conditions whereby the nucleicacid of the composition can be expressed, thereby producing an immuneresponse in the subject.

Also provided is a method of producing an immune response in a subject,comprising administering to the subject a composition comprising afusion polypeptide and a pharmaceutically acceptable carrier and whereinthe fusion polypeptide is a fusion polypeptide comprising a viralchemokine and a human immunodeficiency virus (HIV) antigen, wherein thechemokine can be vMIPI, vMIPII, vMIPIII, or any other viral chemokine,and wherein the HIV antigen can be gp120, gp160, gp41, an active (i.e.,antigenic) fragment of gp120, an active (i.e., antigenic) fragment ofgp160 and/or an active (i.e., antigenic) fragment of gp41, therebyproducing an immune response in the subject.

The present invention also provides a method of producing an immuneresponse in a subject, comprising administering to the subject acomposition comprising a nucleic acid encoding a fusion polypeptidecomprising a viral chemokine and a human immunodeficiency virus (HIV)antigen, wherein the viral chemokine can be vMIPI, vMIPII, or any othervMIPIII viral chemokine, and wherein the HIV antigen can be gp120,gp160, gp41, an active (i.e., antigenic) fragment of gp120, an active(i.e., antigenic) fragment of gp160 and/or an active (i.e., antigenic)fragment of gp41, and a pharmaceutically acceptable carrier, underconditions whereby the nucleic acid can be expressed, thereby producingan immune response in the subject.

In any of the methods provided herein which recite the production of animmune response, the immune response can be humoral and/or an effector Tcell (cellular) immune response, as determined according to methodsstandard in the art.

In another embodiment, the present invention provides a method oftreating a cancer in a subject comprising administering to the subject acomposition comprising a fusion polypeptide of this invention and apharmaceutically acceptable carrier and wherein the fusion polypeptideis a fusion polypeptide comprising vMIPI and human Muc-1, a fusionpolypeptide comprising vMIPII and human Muc-1, or a fusion polypeptidecomprising vMIPIII and human Muc-1, thereby treating a cancer in thesubject.

Additionally provided is a method of treating a cancer in a subject,comprising administering to the subject a composition comprising anucleic acid encoding a fusion polypeptide of this invention and apharmaceutically acceptable carrier and wherein the fusion polypeptideis a fusion polypeptide comprising vMIPI and human Muc-1, or a fusionpolypeptide comprising vMIPII and human Muc-1, or a fusion polypeptidecomprising vMIPIII and human Muc-1, under conditions whereby the nucleicacid of the composition can be expressed, thereby treating a cancer inthe subject.

Further provided is a method of treating or preventing HIV infection ina subject, comprising administering to the subject a compositioncomprising a viral chemokine and a human immunodeficiency virus (HIV)antigen, wherein the viral chemokine can be vMIPI, vMIPII, vMIPIII, orany other viral chemokine, and wherein the HIV antigen can be gp120,gp160, gp41, an active (i.e., antigenic) fragment of gp120, an active(i.e., antigenic) fragment of gp160 and/or an active (i.e., antigenic)fragment of gp41, and a pharmaceutically acceptable carrier, therebytreating or preventing HIV infection in the subject.

In addition, a method of treating or preventing HIV infection in asubject is provided herein, comprising administering to the subject acomposition comprising a nucleic acid encoding a fusion polypeptidecomprising a viral chemokine and a human immunodeficiency virus (HIV)antigen, wherein the viral chemokine can be vMIPI, or any other viralchemokine, and wherein the HIV antigen can be gp120, gp160, gp41, anactive (i.e., antigenic) fragment of gp120, an active (i.e., antigenic)fragment of gp160 and/or an active (i.e., antigenic) fragment of gp41,and a pharmaceutically acceptable carrier, under conditions whereby thenucleic acid can be expressed, thereby treating or preventing HIVinfection in the subject.

Further provided is a method of treating or preventing HIV infection ina subject, comprising administering to the subject a compositioncomprising a fusion polypeptide comprising vMIPI and HIV gp120, a fusionpolypeptide comprising vMIPII and HIV gp120, or a fusion polypeptidecomprising vMIPIII and HIV gp120, and a pharmaceutically acceptablecarrier, thereby treating or preventing HIV infection in the subject.

In addition, a method of treating or preventing HIV infection in asubject is provided herein, comprising administering to the subject acomposition comprising a nucleic acid encoding a fusion polypeptidecomprising vMIPI and HIV gp120, a fusion polypeptide comprising vMIPIIand HIV gp120, or a fusion polypeptide comprising vMIPIII and HIV gp120,and a pharmaceutically acceptable carrier, under conditions whereby thenucleic acid can be expressed, thereby treating or preventing HIVinfection in the subject.

In a further embodiment, the present invention provides a method oftreating a B cell tumor in a subject, comprising administering to thesubject a fusion polypeptide comprising a viral chemokine and a B celltumor antigen, wherein the B cell tumor antigen can be an antibody, asingle chain antibody or an epitope of an idiotype of an antibody, andwherein the viral chemokine can be vMIPI, vMIPII, vMIPIII, or any otherviral chemokine, thereby treating a B cell tumor in the subject.

Also provided is a fusion polypeptide comprising the viral chemokinevMIPI, vMIPII, or vMIPIII and the V3 loop of HIV-1 envelopeglycoprotein, gp120, as well as a fusion protein comprising vMIPI,vMIPII, or vMIPIII and gp160 of HIV-1, a fusion protein comprisingvMIPI, vMIPII, or vMIPIII and gp41 of HIV-1, a fusion protein comprisingvMIPI, vMIPII, or vMIPIII and an active fragment of gp120, a fusionprotein comprising vMIPI, vMIPII, or vMIPIII and an active fragment ofgp160 and a fusion polypeptide comprising vMIPI, vMIPII, or vMIPIII andan active fragment of gp41.

The methods of this invention comprising administering the fusionprotein of this invention to a subject can further comprise the step ofadministering one or more adjuvants, such as an immunostimulatorycytokine to the subject. The adjuvant or adjuvants can be administeredto the subject prior to, concurrent with and/or after the administrationof the fusion protein as described herein.

The subject of the present invention can be any animal in which cancercan be treated by eliciting an immune response to a tumor antigen. In apreferred embodiment, the animal is a mammal and most preferably is ahuman.

To determine the effect of the administration of the fusion polypeptideon inhibition of tumor cell growth in laboratory animals, the animalscan either be pre-treated with the fusion polypeptide and thenchallenged with a lethal dose of tumor cells, or the lethal dose oftumor cells can be administered to the animal prior to receipt of thefusion polypeptide and survival times documented. To determine theeffect of administration of the fusion polypeptide on inhibition oftumor cell growth in humans, standard clinical response parameters canbe analyzed.

To determine the amount of fusion polypeptide which would be aneffective tumor cell growth-inhibiting amount, animals can be treatedwith tumor cells as described herein and varying amounts of the fusionpolypeptide can be administered to the animals. Standard clinicalparameters, as described herein, can be measured and that amount offusion polypeptide effective in inhibiting tumor cell growth can bedetermined. These parameters, as would be known to one of ordinary skillin the art of oncology and tumor biology, can include, but are notlimited to, physical examination of the subject, measurements of tumorsize, X-ray studies and biopsies.

The present invention further provides a method for treating orpreventing HIV infection in a human subject, comprising administering tothe subject an HIV replication-inhibiting amount of the viralchemokine/HIV antigen fusion polypeptide of this invention. As usedherein, “a replication-inhibiting amount” is that amount of fusionpolypeptide which produces a measurable humoral and/or effector T cell(cellular) immune response in the subject against the viral antigen, asdetermined by standard immunological protocols, resulting in theinhibition of HIV replication in cells of the subject, as determined bymethods well known in the art for measuring HIV replication, such asviral load measurement, which can be determined by quantitative PCR(QPCR) and branched DNA (bDNA) analysis; reverse transcriptase activitymeasurement, in situ hybridization, Western immunoblot, ELISA and p24gag measurement (87,88,89,90,91). The fusion polypeptide can beadministered to the subject in varying amounts and the amount of thefusion polypeptide optimally effective in inhibiting HIV replication ina given subject can be determined as described herein.

The fusion polypeptide of this invention can be administered to thesubject orally or parenterally, as for example, by intramuscularinjection, by intraperitoneal injection, topically, transdermally,injection directly into the tumor, or the like, although subcutaneousinjection is typically preferred. Immunogenic, tumor cell growthinhibiting and HIV replication inhibiting amounts of the fusionpolypeptide can be determined using standard procedures, as described.Briefly, various doses of the fusion polypeptide are prepared,administered to a subject and the immunological response to each dose isdetermined (92). The exact dosage of the fusion polypeptide will varyfrom subject to subject, depending on the species, age, weight andgeneral condition of the subject, the severity of the cancer or HIVinfection that is being treated, the particular antigen being used, themode of administration, and the like. Thus, it is not possible tospecify an exact amount. However, an appropriate amount may bedetermined by one of ordinary skill in the art using only routinescreening given the teachings herein.

Generally, the dosage of fusion protein will approximate that which istypical for the administration of vaccines, and typically, the dosagewill be in the range of about 1 to 500 μg of the fusion polypeptide perdose, and preferably in the range of 50 to 250 μg of the fusionpolypeptide per dose. This amount can be administered to the subjectonce every other week for about eight weeks or once every other monthfor about six months. The effects of the administration of the fusionpolypeptide can be determined starting within the first month followingthe initial administration and continued thereafter at regularintervals, as needed, for an indefinite period of time.

For oral administration of the fusion polypeptide of this invention,fine powders or granules may contain diluting, dispersing, and/orsurface active agents, and may be presented in water or in a syrup, incapsules or sachets in the dry state, or in a nonaqueous solution orsuspension wherein suspending agents may be included, in tablets whereinbinders and lubricants may be included, or in a suspension in water or asyrup. Where desirable or necessary, flavoring, preserving, suspending,thickening, or emulsifying agents may be included. Tablets and granulesare preferred oral administration forms, and these may be coated.

Parenteral administration, if used, is generally characterized byinjection. Injectables can be prepared in conventional forms, either asliquid solutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. A morerecently revised approach for parenteral administration involves use ofa slow release or sustained release system, such that a constant levelof dosage is maintained. See, e.g., U.S. Pat. No. 3,710,795, which isincorporated by reference herein.

For solid compositions, conventional nontoxic solid carriers include,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose,magnesium carbonate, and the like. Liquid pharmaceutically administrablecompositions can, for example, be prepared by dissolving, dispersing,etc. an active compound as described herein and optional pharmaceuticaladjuvants in an excipient, such as, for example, water, saline, aqueousdextrose, glycerol, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like, for example, sodium acetate, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, etc. Actualmethods of preparing such dosage forms are known, or will be apparent,to those skilled in this (84).

The present invention also provides a method for producing single chainantibodies against tumor antigens comprising producing a fusionpolypeptide comprising a viral chemokine region and a region comprisinga tumor antigen; immunizing animals with an amount of the fusionpolypeptide sufficient to produce a humoral immune response to thefusion polypeptide; isolating spleen cells expressing immunoglobulinspecific for the fusion polypeptide; isolating the immunoglobulinvariable genes from the spleen cells; cloning the immunoglobulinvariable genes into an expression vector; expressing the immunoglobulinvariable genes in a bacteriophage; infecting E. coli cells with thebacteriophage; isolating bacteriophage from the E. coli cells whichexpress the immunoglobulin variable genes and isolating theimmunoglobulin variable gene products for use as single chainantibodies.

The viral chemokine-scFv fusion proteins described herein would bebetter targets than tumor cells or purified tumor antigen peptides forantibody selection approaches such as phage displayed scFv production.For example, there are two ways to produce specific Fv displayed on thesurface of phage: (1) Immunize mice with tumor cells; isolateimmunoglobulin variable fragment genes from spleen cells by RT/PCR;clone the genes into bacteriophage in frame with genes coding phagesurface proteins (e.g., major coat protein subunits gpVIII or gp III ofthe filamentous bacteriophage) (93,94); and (2) Construct semisyntheticantibody libraries by PCR as described (95). The specific phageproducing scFv are selected by several rounds of binding elution andinfection in E. coli, using biotin labeled chemokine-tumor antigen(e.g., Muccore). The biotin enables selection of high affinityscFv-phage through binding to streptavidin conjugated magnetic beads.This approach provides simple, fast and efficient production of specificanti-tumor epitope scFv.

As described herein, the present invention also provides a nucleic acidwhich encodes a fusion polypeptide of this invention and a vectorcomprising a nucleic acid which encodes a fusion polypeptide of thisinvention, either of which can be in a pharmaceutically acceptablecarrier. Such nucleic acids and vectors can be used in gene therapyprotocols to treat cancer as well as to treat or prevent HIV infectionin a subject.

Thus, the present invention further provides a method of treating acancer in a subject diagnosed with a cancer comprising administering thenucleic acid of this invention to a cell of the subject under conditionswhereby the nucleic acid is expressed in the cell, thereby treating thecancer.

A method of treating a B cell tumor in a subject diagnosed with a B celltumor is also provided, comprising administering the nucleic acid ofthis invention, encoding a viral chemokine and an antibody or fragmentthereof, in a pharmaceutically acceptable carrier, to a cell of thesubject, under conditions whereby the nucleic acid is expressed in thecell, thereby treating the B cell tumor.

The methods of this invention comprising administering nucleic acidencoding the fusion protein of this invention to a subject can furthercomprise the step of administering a nucleic acid encoding an adjuvantsuch as an immunostimulatory cytokine to the subject, either before,concurrent with or after the administration of the nucleic acid encodingthe fusion protein, as described herein.

The nucleic acid can be administered to the cell in a virus, which canbe, for example, adenovirus, retrovirus and adeno-associated virus.Alternatively, the nucleic acid of this invention can be administered tothe cell in a liposome. The cell of the subject can be either in vivo orex vivo. Also, the cell of the subject can be any cell which can take upand express exogenous nucleic acid and produce the fusion polypeptide ofthis invention. Thus, the fusion polypeptide of this invention can beproduced by a cell which secretes it, whereby it binds a viral chemokinereceptor and is subsequently processed by an antigen presenting cell andpresented to the immune system for elicitation of an immune response.Alternatively, the fusion polypeptide of this invention can be producedin an antigen presenting cell where it is processed directly andpresented to the immune system.

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The nucleic acids of this invention can be introduced intothe cells via any gene transfer mechanism, such as, for example,virus-mediated gene delivery, calcium phosphate mediated gene delivery,electroporation, microinjection or proteoliposomes. The transduced cellscan then be infused (e.g., in a pharmaceutically acceptable carrier) ortransplanted back into the subject per standard methods for the cell ortissue type. Standard methods are known for transplantation or infusionof various cells into a subject.

For in vivo methods, the nucleic acid encoding the fusion protein can beadministered to the subject in a pharmaceutically acceptable carrier asdescribed herein.

In the methods described herein which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), the nucleic acids of the presentinvention can be in the form of naked DNA or the nucleic acids can be ina vector for delivering the nucleic acids to the cells for expression ofthe nucleic acid to produce the fusion protein of this invention. Thevector can be a commercially available preparation, such as anadenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec,Canada). Delivery of the nucleic acid or vector to cells can be via avariety of mechanisms. As one example, delivery can be via a liposome,using commercially available liposome preparations such as LIPOFECTIN,LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen,Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison,Wis.), as well as other liposomes developed according to proceduresstandard in the art. In addition, the nucleic acid or vector of thisinvention can be delivered in vivo by electroporation, the technologyfor which is available from Genetronics, Inc. (San Diego, Calif.) aswell as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp.,Tucson, Ariz.).

Vector delivery can also be via a viral system, such as a retroviralvector system which can package a recombinant retroviral genome (seee.g., 96,97). The recombinant retrovirus can then be used to infect andthereby deliver to the infected cells nucleic acid encoding the fusionpolypeptide. The exact method of introducing the exogenous nucleic acidinto mammalian cells is, of course, not limited to the use of retroviralvectors. Other techniques are widely available for this procedureincluding the use of adenoviral vectors (98), adeno-associated viral(AAV) vectors (99), lentiviral vectors (100), pseudotyped retroviralvectors (101). Physical transduction techniques can also be used, suchas liposome delivery and receptor-mediated and other endocytosismechanisms (see, for example, 102). This invention can be used inconjunction with any of these or other commonly used gene transfermethods.

Various adenoviruses may be used in the compositions and methodsdescribed herein. For example, a nucleic acid encoding the fusionprotein can be inserted within the genome of adenovirus type 5.Similarly, other types of adenovirus may be used such as type 1, type 2,etc. For an exemplary list of the adenoviruses known to be able toinfect human cells and which therefore can be used in the presentinvention, see Fields, et al. (103). Furthermore, it is contemplatedthat a recombinant nucleic acid comprising an adenoviral nucleic acidfrom one type adenovirus can be packaged using capsid proteins from adifferent type adenovirus.

The adenovirus of the present invention is preferably renderedreplication deficient, depending upon the specific application of thecompounds and methods described herein. Methods of rendering anadenovirus replication deficient are well known in the art. For example,mutations such as point mutations, deletions, insertions andcombinations thereof, can be directed toward a specific adenoviral geneor genes, such as the E1 gene. For a specific example of the generationof a replication deficient adenovirus for use in gene therapy, see WO94/28938 (Adenovirus Vectors for Gene Therapy Sponsorship) which isincorporated herein in its entirety.

In the present invention, the nucleic acid encoding the fusion proteincan be inserted within an adenoviral genome and the fusion proteinencoding sequence can be positioned such that an adenovirus promoter isoperatively linked to the fusion protein nucleic acid insert such thatthe adenoviral promoter can then direct transcription of the nucleicacid, or the fusion protein insert may contain its own adenoviralpromoter. Similarly, the fusion protein insert may be positioned suchthat the nucleic acid encoding the fusion protein may use otheradenoviral regulatory regions or sites such as splice junctions andpolyadenylation signals and/or sites. Alternatively, the nucleic acidencoding the fusion protein may contain a different enhancer/promoter(e.g., CMV or RSV-LTR enhancer/promoter sequences) or other regulatorysequences, such as splice sites and polyadenylation sequences, such thatthe nucleic acid encoding the fusion protein may contain those sequencesnecessary for expression of the fusion protein and not partially ortotally require these regulatory regions and/or sites of the adenovirusgenome. These regulatory sites may also be derived from another source,such as a virus other than adenovirus. For example, a polyadenylationsignal from SV40 or BGH may be used rather than an adenovirus, a human,or a murine polyadenylation signal. The fusion protein nucleic acidinsert may, alternatively, contain some sequences necessary forexpression of the nucleic acid encoding the fusion protein and deriveother sequences necessary for the expression of the fusion proteinnucleic acid from the adenovirus genome, or even from the host in whichthe recombinant adenovirus is introduced.

As another example, for administration of nucleic acid encoding thefusion protein to an individual in an AAV vector, the AAV particle canbe directly injected intravenously. The AAV has a broad host range, sothe vector can be used to transduce any of several cell types, butpreferably cells in those organs that are well perfused with bloodvessels. To more specifically administer the vector, the AAV particlecan be directly injected into a target organ, such as muscle, liver orkidney. Furthermore, the vector can be administered intraarterially,directly into a body cavity, such as intraperitoneally, or directly intothe central nervous system (CNS).

An AAV vector can also be administered in gene therapy procedures invarious other formulations in which the vector plasmid is administeredafter incorporation into other delivery systems such as liposomes orsystems designed to target cells by receptor-mediated or otherendocytosis procedures. The AAV vector can also be incorporated into anadenovirus, retrovirus or other virus which can be used as the deliveryvehicle.

As described above, the nucleic acid or vector of the present inventioncan be administered in vivo in a pharmaceutically acceptable carrier. By“pharmaceutically acceptable” is meant a material that is notbiologically or otherwise undesirable, i.e., the material may beadministered to a subject, along with the nucleic acid or vector,without causing any undesirable biological effects or interacting in adeleterious manner with any of the other components of thepharmaceutical composition in which it is contained. The carrier wouldnaturally be selected to minimize any degradation of the activeingredient and to minimize any adverse side effects in the subject, aswould be well known to one of skill in the art.

The mode of administration of the nucleic acid or vector of the presentinvention can vary predictably according to the disease being treatedand the tissue being targeted. For example, for administration of thenucleic acid or vector in a liposome, catheterization of an arteryupstream from the target organ is a preferred mode of delivery, becauseit avoids significant clearance of the liposome by the lung and liver.

The nucleic acid or vector may be administered orally as describedherein for oral administration of the fusion polypeptides of thisinvention, parenterally (e.g., intravenously), by intramuscularinjection, by intraperitoneal injection, transdermally,extracorporeally, topically or the like, although intravenousadministration is typically preferred. The exact amount of the nucleicacid or vector required will vary from subject to subject, depending onthe species, age, weight and general condition of the subject, theseverity of the disorder being treated, the particular nucleic acid orvector used, its mode of administration and the like. Thus, it is notpossible to specify an exact amount for every nucleic acid or vector.However, an appropriate amount can be determined by one of ordinaryskill in the art using only routine experimentation given the teachingsherein (84).

As one example, if the nucleic acid of this invention is delivered tothe cells of a subject in an adenovirus vector, the dosage foradministration of adenovirus to humans can range from about 10⁷ to 10⁹plaque forming units (pfu) per injection, but can be as high as 10¹² pfuper injection (104,105). Ideally, a subject will receive a singleinjection. If additional injections are necessary, they can be repeatedat six month intervals for an indefinite period and/or until theefficacy of the treatment has been established.

Parenteral administration of the nucleic acid or vector of the presentinvention, if used, is generally characterized by injection. Injectablescan be prepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution or suspension in liquidprior to injection, or as emulsions. A more recently revised approachfor parenteral administration involves use of a slow release orsustained release system such that a constant dosage is maintained. See,e.g., U.S. Pat. No. 3,610,795, which is incorporated by reference hereinin its entirety.

The present invention is more particularly described in the followingexamples which are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

EXAMPLES Example 1 Materials and Methods

Mice and tumor. C3H/HeN female mice 6 to 12 weeks of age are obtainedfrom the Animal Production Area of the National CancerInstitute-Frederick Cancer Research and Development Center (NCI-FCRDC,Frederick, Md.). The cell line 38c13 is a carcinogen-induced murine Bcell tumor cell line (125). The 38c13 tumor cell secretes and expressesIgM(κ) on the cell surface and is MHC class I positive but class IInegative. 38c13 cells from a common frozen stock are passaged in vitro 3days before use in RPMI 1640 supplemented with 100 U/ml of penicillinand streptomycin, 2×10⁻⁵M 2-mercaptoethanol and heat inactivated 10%fetal bovine serum (BioWhitaker).

Construction of expression vectors. Two types of expression systems areused to produce scFv and scFv fusions. In one system, nucleic acidencoding the fusion protein is expressed in a modified pet11d vector(Stratagene) and purified from inclusion bodies of E. coli. In thesecond system, the nucleic acid encoding the fusion polypeptide iscloned into a pCMVE/AB (Arya Biragyn) vector under regulatory elementsof the early promoter and enhancer of CMV and expressed in the epidermisof mice as a naked DNA vaccine.

Fv fragments are cloned from two different B cell lymphomas, 38C13 andA20, respectively (106,107) by RT/PCR and produced as recombinant fusionpeptides with either IP-10, respectively designated as IP10scFv38 andIP10scFv20A, or MCP3scFv38 and MCP3scFv20A. Specifically, lymphomaspecific Vh and VI fragments are cloned by RT/PCR techniques as singlechain antibody from total RNA of 38c13 and A20 tumor cells, designatedscFv38 and scFv20A respectively, using the following primers.

PRVh-5′: PRV_(H)38-5′: (SEQ ID NO: 4) CTCGAGG TGAAGCTGGTGGAGTCTGGAPRVh-3′: PRV_(H)38-3′: (SEQ ID NO: 5) AGAGGAGACTGTGAGAGTGGTGCCTTPRVl-5′: PRV_(L)38-5′: (SEQ ID NO: 6) GACATCCAGATGACACAGTCTCCAPRVl-3′: PRV_(L)38-3′: (SEQ ID NO: 7)GGATCCTTTTATTTCCAGCTTGGTCCCCCCTCCGAA PRV_(H)20A-5′: (SEQ ID NO: 8)CCATGGTCCAAC TGCAGCAGTCAGGGCCTGAC PRV_(H)20A-3′: (SEQ ID NO: 9)TGAGGAGACTGTGAGTTCGGTACCTT GGCC PRV_(L)20A-5′: (SEQ ID NO: 10)GATGTTGTGATGACGCAGACTCCACTC PRV_(L)20A-3′: (SEQ ID NO: 11)GGATCCTTTGACTTCCAGCTTTGTGCCTCCA

The resulting scFv contained a (Gly₄Ser)₃ linker and is cloned into theexpression vector pET11d, which is modified to fuse in frame with c-mycand the His tag peptide sequences, followed by an amber stop codon. Theresulting scFv contains a 17 a.a. residue linker, GGGGSGGGGSGGGGSGS(Gly₄Ser)₃GlySer (SEQ ID NO:12) (108).

Constructs for the nDNA vaccination are fused in frame to a leadersequence of vMIPI in pCMVE/AB to enable secretion. The carboxy-terminusof scFv is fused in frame with the tag sequence encoding c-myc peptideand six His residues, respectively: GGA TCC GCA GAA GAA CAG AAA CTG ATCTCA GAA GAG GAT CTG GCC CAC CAC CAT CAC CAT CAC TAA CCCGGG (SEQ IDNO:13). Genes for the mature sequence of viral chemokines vMIPI, vMIPII,and vMIPII are cloned by RT/PCR technique from cell lines infected withHHV-8 as would be known in the art, and fused in frame with sFvutilizing suitable primers to form vMIPIsFv38, vMIPIIsFv38 andvMIPIIIsFv38.

VMIPI, vMIPII, vMIPIII or control viral epitope (PreS2 and DomA) fusionsare made by fusing them to amino-terminus of scFv through a short spacersequence: 5′ GAA TTC AAC GAC GCT CAG GCG CCG AAG AGT CTC GAG 3′ (SEQ IDNO:14), encoding the amino acid sequence: EFNDQAPKSLE (SEQ ID NO:15).Two unique restriction endonuclease sites are introduced at the ends ofthe space to facilitate cloning: EcoRI at the 5′ end (underlined) andXhoI at the 3′ end (underlined). All constructs are verified by DNAdideoxy-sequencing method, using T7 SEQUENASE kit (Amersham).

vMIPI, vMIPII, and vMIPIII chemokines are cloned into the scFv38expression vector through NcoI and XhoI restriction sites. The resultingfusion nucleic acid contains the viralchemokine gene ligated to the5′-end of the scFv38 gene and separated with a short spacer sequence, asdescribed above.

Bacterial expression and scFv purification. The recombinant proteins areexpressed in BL21(DE3) cells (InVitrogen) as inclusion bodies after 8hours of induction in Super-Broth with 0.8 mM IPTG in the presence of150 μg/ml carbenicillin and 50 μg/ml ampicillin at 30° C. vMIPI-scFv38and vMIPII-scFv38 are purified from the inclusion bodies with a modifiedmethod (110). Briefly, inclusion bodies, denatured in 6M GuHCl, 100 mMNaH₂PO₄, 10 mM Tris-HCl, pH 8.0, are reduced in 0.3M DTE and refolded ata concentration of 80 μg/ml in the refolding solution (Tris-HCl, pH 8.0,0.5M arginine-HCL, 4 mM GSSG and 2 mM EDTA) for 72 hours at 10° C. Therefolded solution is dialyzed in 100 mM Urea and 20 mM tris-HCl, pH 7.4and the recombinant protein is purified by binding to heparin-sepharoseresins (Pharmacia, Biotech, Uppsala, Sweden). The integrity and purityof the recombinant protein is tested by PAGE gel electrophoresis inreducing conditions and by Western blot hybridization with mAb 9E10. Thepurification yields 2-20 mg/l of the soluble protein with greater than90% purity.

Results

Purified fusion polypeptide is tested for the ability to inhibit bindingof native IgM 38c13 (Id38), as compared to positive sera from miceimmunized with Id38-KLH. ELISA plates are coated with 10 g/ml Id38, thenwells are incubated with anti-Id38 positive sera (1:500) and titratedamounts of scFv. Id38 (10 μg/ml) and either vMIPIscFv20 or vMIPIIscFv20(vMIPI, vMIPII or vMIPIII fused to an irrelevant scFv) are used aspositive and negative control samples, respectively.

Recombinant fusion proteins purified from E. coli are characterized forproper idiotype folding by their ability to inhibit 38c13 IgM binding toa monoclonal (SIC5 mAb) or polyclonal anti-idiotypic sera, in order todetermine if vMIPI, vMIPII, or vMIPIII fusions interfere with the properconformation of scFv38. Next, receptor binding experiments are used todetermine whether either vMIPI, vMIPII, or vMIPIII fused scFv, but notcontrol viral epitope DomA fused scFv38 (DomAscFv38), bind to theirchemokine receptors. Accordingly, vMIPIsFv38, vMIPIIsFv38, andvMIPIIIsFv38 are each tested for their ability to bind to CCR5 and CXCR4transfected cell lines as known in the art. vMIPIIsFv38 does bind toCCR5 and CXCR4 transfected cell lines, as evidenced by the ability ofvMIPIIsFv38 to displace labeled MIP1.

Furthermore, vMIPIsFv38, vMIPIIsFv38, and vMIPIIIsFv38 are tested fortheir ability to induce chemotaxis of THP-1 cells. As expected,vMIPIIsFv38 does not induce chemotaxis, while vMIPIIsFv38 does retainchemotaxis activity.

In vivo immunization and tumor protection. To test the ability ofvMIPIsFv38, vMIPIIsFv38, and vMIPIIisFv38 to render self tumor antigen,sFv38, immunogenic when immunized as genetic vaccine in mice, ten miceper group are gene-gun immunized with plasmid encoding fusions with theviral chemokine fusions pvMIPIsFv38, pvMIPIIsFv38, and pvMIPIIIsFv38 inorder to demonstrate whether these fusions can target APC in vitro. As acontrol, mice are immunized with the similar DNA constructs but encodingmutated chemokine fusions pvMIPIDsFv38, pvMIPIIDsFv38, andpvMIPIIIDsFv38. The viral chemokines vMIPI and vMIPII induce significantanti-idiotype specific antibodies compared to the prototype Ig38-KLHprotein vaccine. These results are in distinct contrast with the lack ofany anti-idiotypic antibody response after immunization with fusionswith mutated chemokines. Thus, the non-immunogenic sFv is renderedimmunogenic by viral chemokines, and the response correlates with theability to induce chemotaxis of immature DC and other APC.

Groups of ten mice immunized with viral chemokine or control plasmidsare challenged with 20-fold lethal dose of tumor two weeks after thelast of three serial immunizations. Mice immunized with viral chemokinemutants pvMIPIDsFv38 and pvMIPIIDsFv38, similarly to mice immunized withcorresponding active chemokines fused with sFvA20 from irrelevantlymphoma A20 sFv or PBS, do not survive. In contrast, pvMIPIsFv38 orpvMIPIIsFv38 immunized mice demonstrate statistically significantsurvival. The survival closely correlates with the presence offunctionally intact or active viral chemokines which can act on immatureDC or other APC via differentially expressing their receptors. AlthoughGen-gun bombarded DNA does target a variety of skin cells including skinAPC and LC, it is not sufficient to render self-tumor antigen (sFv)immunogenic. Instead, physical linkage of sFv with viral chemokinemoieties is required. No positive humoral response or survival isdetected in mice immunized with DNA plasmids expressing free unlinkedviral chemokine and sFv38.

Thus, the animal experiments clearly correlate with functional data fromin vitro studies. Id-specific responses and tumor immunity is detectedonly when the viral chemokine moiety in the fusion retains thefunctional properties of the respective viral chemokine, while noimmunity is observed when the viral chemokine is replaced with a mutant,functionally inactive chemokine. Furthermore, induction of chemotaxis tothe site of vaccine injection or production is not sufficient togenerate, as demonstrated by the lack of humoral and anti-tumor immunityin mice immunized with viral chemokine alone or with either a mixture offree, unlinked viral chemokine and sFv or viral chemokine fused with anirrelevant sFv.

Example 2

Recent experiments also demonstrate that vMIP-II elicits antigenspecific responses against human breast cancer Muc-1 antigen and envprotein of HIV-1.

Six- to nine-week old female C3H/HeN mice are immunizedintraperitoneally (i.p.) with 100 to 200 μg of the soluble protein inPBS and control immunogen Id38-KLH two times at two week intervals orare shaved and immunized by Accell gene delivery device (Agracetus,Inc., Middleton, WN) with 1 μg gold particles carrying 1-3 μg plasmidDNA. Sera are collected by orbital bleeding two weeks after eachvaccination. Serum anti-idiotypic (anti-Id) antibody levels are testedas described (111) over microtiter plates coated with 10 μg/ml nativeIgM 38c13. Two weeks after the last immunization, mice are inoculatedwith 2000 38c13 tumor cells i.p. Survival is determined, andsignificance with the respect to time to death, is assessed using BMDPIL software (BMDP statistical software, Los Angeles). Mice are observeddaily for any signs of toxicity and date of death and animalssurviving >80 days after tumor challenge are killed and reported aslong-term survivors.

Mice are immunized either with a plasmid coding for a vMIPIsFv38 fusion,a vMIPIIsFv38 fusion, a vMIPIIIsFv38 fusion, or a mixture of DNAconstructs expressing unlinked scFv38 and vMIPIscFv20A(scFv38D+vMIPIscfv20AD).

Ten mice per group are immunized with two types of scFv38 fused tovMIPI, vMIPII or vMIPIII, differing only in orientation of variablegenes in scFv. Control mice receive IgM-KLH (Id38-KLH) and vMIP fusionto A20 lymphoma scFv (IP10scFv20A). Ten mice per group are immunizedi.d. with plasmid coding either for viral chemokine fusion vaccine(vMIPIscFv38D, vMIPIIscFv38D, or MIPIIIscFv38D), or free scFv (scFv38D),or viral epitope preS2 fused scFv (PreS2scFv38D).

Effector CD8⁺ and CD4⁴⁺ cells are depleted two weeks after the lastimmunization with three i.p. injections of 400 μg α-CD8 mAb 53.6.72, orα-CD4 mAb GK1.5 (both ammonium sulfate purified ascites, BiologicalResource Branch, NCI-FCRDC) (32,34), or control rat IgG (Sigma). Controlmice are immunized with plasmid expressing vMIPI, vMIPII, or vMIPIIIfused to A20 scFv (MCP3scFv20AD).

Ten Balb/C mice per group are immunized i.p. twice with 100 μg of vMIPI,vMIPII, or vMIPIII fused with scFv20A protein in PBS (vMIPIscFv20A andvMIPIIscFv20A, respectively) and challenged i.p. with 10⁵ A20 tumorcells. To determine the role of free versus linked chemokine,vMIPIscFv20A is co-injected with vMIPII fused to an irrelevant scFv38(vMIPIscFv20A+vMIPIIscFv38). Control mice are immunized with A20 IgM-KLH(Id20A-KLH).

Immunoassays and serum anti-idiotypic antibody. The assessments forcorrect folding of purified scFv38 and fusion scFv38 are determined byELISA with mAbs and by inhibition assay with Id38-KLH sera (immunizedwith native IgM 38c13 conjugated to KLH). Briefly, microtiter plates(Nunc, Naperville, Ill.) are coated overnight at 4° C. with 10 μg/mlanti-c-myc mAb 9E10 in carbonate buffer (50 mM NaHCO₃, pH 9.0). Thewells are blocked with 5% nonfat dry milk in PBS for 30 min. Plates arewashed in 0.05% Triton X-100 in PBS, and serially diluted scFv (startingfrom 10 μg/ml in 50 μl 2% BSA/PBS) is applied, after which plates areincubated 40 min at room temperature. After washing, the wells areincubated with 50 μl of 1:300 diluted biotinylated anti-Id38 mAb in 2%BSA/PBS for 30 min at room temperature. Wells are washed and incubatedwith streptavidin-HRP conjugate (1:5000) in 2% BSA/PBS for 30 min atroom temperature. Then, wells are washed and incubated with ABTSperoxidase substrate (KPL, Gaithersburg, Md.) and the absorbance at 405nm is measured.

Inhibition assays are performed as described above, except plates arecoated with 10 μg/ml of native IgM 38c13, then wells are incubated for30 min at room temperature with a 1:2 dilution of positive Id38-KLH seramixed with serially diluted purified scFv proteins starting from 50μg/ml in 2% BSA/PBS. The bound antibodies from the sera are assayed byincubating wells for 30 min at room temperature with anti-mouse IgG-HRPmAb (Jackson).

Serum anti-idiotypic (anti-Id) antibody levels are tested as described(37). Briefly, mouse serum is serially diluted over microtiter platescoated with 10 μg/ml native IgM 38c13. Binding of antibodies in theserum to 38c13 IgM is detected by goat anti-mouse IgG-HRP. Serum anti-Idantibody levels are quantitated by comparing sera titration curves witha standard curve obtained with a known concentration of a mixture ofpurified monoclonal anti-Id antibodies. Antibody levels are expressed ing/ml of serum for individual mice. In each ELISA, sera obtained frommice immunized with control IgM-KLH are included as negative controls.Such sera never showed any titration binding activity on Id-38c13.

In vitro and in vivo chemotaxis assays. Single cell suspensions areprepared from spleens of untreated C3H/HeJ mice. Murine T cellenrichment columns (R&D System, Minneapolis, Minn.) are then used toprepare a purified murine T cell population via high-affinity negativeselection according to the manufacturer's instructions. The isolationprocedure typically yields over 89% CD3⁺ T cells, as determined by FACSanalysis. T cell migration in vitro is assessed by 48-wellmicrochemotaxis chamber technique. Briefly, a 26 μl aliquot of therecombinant scFv fusion protein serially diluted in the chemotaxismedium (RPMI 1640, 1% BSA, 25 mM HEPES) is placed in the lowercompartment and 50 μl of cell suspension (5×10⁶ cells/ml) is placed inthe upper compartment of the chamber. The two compartments are separatedby a polycarbonate filter (5 μm pore size; Neuroprobe, Cabin John, M D)coated with 10 μg/ml of fibronectin (Sigma, St. Luis, Mo.) and incubatedovernight at 4° C. or for 2 hours at 37° C. The chemotaxis assay isperformed at 37° C. for 2 hours. Then the filter is removed, fixed andstained with Diff-Quik (Harlew, Gibbstown, N.J.). The number of migratedcells in three high power fields (400×) is counted by light microscopyafter coding the samples. The results are expressed as the mean ±SEvalue of the migration in triplicate samples.

T cell migration in vitro is assessed by the 48-well micro chemotaxischamber technique as described (112). Single cell suspensions areprepared from spleens of untreated C3H/HeJ mice. Murine T cellenrichment columns (R&D System, Minneapolis, Minn.) are then used toprepare a purified murine T cell population via high-affinity negativeselection according to the manufacturer's instruction. The isolationprocedure typically yields over 89% CD3⁺ T cells, as determined by FACSanalysis.

In order to test in vivo effects on cell accumulation, C3H/HeN mice areinjected s.c. with a single 10 μg dose of scFv fusion proteins. Portionsof the skin from the site of injection are removed 72 hours after theinjection, fixed in 10% neutral buffered formalin, embedded in paraffin,sectioned at 5 μm and stained with hematoxylin and eosin (H&E). Slidesare evaluated microscopically without knowledge of the experimentaltreatment.

In vivo cellular infiltration into murine skin. The numbers of PMN andmononuclear (MN) cells infiltrated into murine skin are graded asfollowing:—, no significant lesion; 1, mild; 2, moderate; 3 severe; F,focal; MF, multi focal. Mice are injected with 10 μg of vMIPIscFv38,vMIPIIscFv38, vMIPIIscFv38, preS2scFv38 (N18), or PBS, subcutaneously.After 72 h, the injection site is excised and examined histologically oncoded slides to determine the extent of infiltration. The amount ofendotoxin injected with samples is 0.5-1 units.

Chemokine binding assay and confocal microscopy. Chemokine bindingassays are performed using laser confocal microscopy (113). Purified Tcells or spleen cells from C3H mice are used at ˜1×10⁶ per ml and areincubated with 100 nM viral chemokine-scFv (vMIPIscFv38, vMIPIIscFv38,or vMIPIIscFv38), or control viral epitope-scFv for 1 hour at 37° C. Forthe ligand competition assay, 100 nM chemokine-scFv is incubated with500 nM of the corresponding chemokine (IP-10 or MCP-3). Samples arewashed 2× in PBS and fixed in suspension with 2% paraformaldehyde. Thesamples are incubated at RT for 15 min. Slides containing the samplesare incubated in 9E10 anti c-myc mAb primary antibody at a 1:50 dilutionin wash buffer (0.25% gelatin, 0.15% saponin, 1% goat serum in PBS).Slides are then incubated with goat anti-mouse IgG F(ab′)2-FITC(Boehringer-Mannheim) at a 1:50 dilution for 30 min at RT in ahumidified chamber. Slides are washed 3×5 min in 0.25% gelatin, 0.15%saponin in TBS. Slides are then incubated for 10 min in a 1:100 dilutionof DAPI, washed 2× briefly in TBS, then 1× briefly in dH₂O, air-driedand mounted using aqueous mounting medium appropriate forimmunofluorescence (Gel/Mount, Biomeda).

The traditional approach to enhance immunogenicity by cross linking toKLH is not effective. Several different approaches are used for theproduction of single chain antibody fragments from 38c13 cells (scFv38)in E. coli. Yield of scFv38 differ significantly depending on the methodused. Production of scFv38 through a secretory path using a PelB leadersequence as a native protein is least efficient. The problem is solvedwhen scFv38 is produced as insoluble “inclusion” bodies, which yieldabout 2-8 mg of refolded scFv per liter of the batch culture withgreater than 90% purity. Folding properties of the produced scFv38 aremonitored by either (i) inhibition assay with native Id38; or (ii)modified ELISA assay where scFv38 is captured through an anti-c-myc tagand detected with the biotinylated monoclonal anti-Id38 antibody(anti-Id38 mAb does not recognize linear or incorrectly folded epitope).These experiments demonstrate that scFv38, but not irrelevant scFv20A,specifically binds to anti-Id38c mAb and inhibits binding of the nativeId38c to anti-Id38c mAb, 50% binding inhibition by 10-15 fold excess ofscFv38. In addition, positive sera from Id38c-KLH immunized micespecifically recognizes purified scFv38. These data indicate thatpurified scFv38 is folded correctly and imitates the idiotype of thenative antibody (Id38c) of B cell lymphoma 38c13.

Immunization-experiments showed that scFv38, similarly to the nativeId38c IgM, is a poor immunogen. Attempts were made to convert scFv38into a potent immunogen by chemical cross linking with KLH, in analogyto the native Id38c. However, in contrast to Id38-KLH, i.p.immunizations of syngeneic mice with 100 μg of scFv38-KLH did not elicitany anti-Id38c specific antibody response. This inability to induceanti-Id38 response correlates with the loss of ability to affect bindingof anti-Id38 mAb (SIC5) to Id38c by samples containing scFv38-KLH, whilea control sample of an equimolar mixture of non-cross linked scFv38 andKLH (scFv38+KLH) inhibited anti-Id38/Id38c binding similarly to purescFv38. These data indicate that a fragile Id conformation of scFv38 isremoved by KLH cross linking and that this traditional approach is notapplicable for the enhancement of immunogenicity of scFv38.

Design and Production of Chemokine Fused scFv38. Viral MIPI, MIME andvMIPIII are subcloned from cell lines infected with HHV-8 by RT/PCRusing specific primers as described herein and inserted in frame infront of the scFv38 DNA sequence. The resulting fusion gene isdesignated as vMIPIscFv38, vMIPIIscFv38, and vMIPIIIscFv38,respectively. In order to evaluate input of the immunoglobulin V chainspecific orientation, two variants of fusion chemokine-scFv genes aredesigned, one containing a V_(H)-V_(L) and one containing a V_(L)-V_(H)sequence, respectively designated as scFv38 MH and scFv38(INV)MH.

All fusion proteins used in these experiments are purified frominclusion bodies of E. coli, solubilized and refolded as describedherein. A spacer sequence, as described herein, is introduced into thechemokine fusion proteins and correct folding is tested for eachrecombinant protein.

The ability of vMIPIscFv38, vMIPIIscFv38 and vMIPIIscFv38 proteins toinduce chemotaxis in vivo in C3H/HeN mice is also tested. Mice are s.c.injected once with 10 μg of the fusion protein and after 72 hours, theskin around the site of injection is removed and analyzed as describedherein.

Production of fusion polypeptides comprising a human chemokine and ahuman tumor antigen or HIV antigen. To produce the fusion polypeptidesof the present invention which comprise a viral chemokine region and ahuman tumor antigen region or HIV antigen region, the followingprocedures are carried out: Tumor or viral antigen is cloned by PCR orRT/PCR from DNA or RNA of biopsy cells of a patient, using specificprimer. The primers are made using standard methods for selecting andsynthesizing primer sequences from analysis of known sequences of thegenes of interest (e.g., from GenBank, Kabat Ig sequence database andother available genetic databases, as are known in the art). Forexample, lymphoma or myeloma-specific scFv is cloned by RT/PCR from thenucleic acid from a patient's lymphoma or myeloma biopsy cells or fromnucleic acid from hybridoma cells expressing the patient'simmunoglobulin. Several sets of primers are used to clone human variable(V) genes based on GenBank and Kabat IG sequence data. As in cloningmurine scFv, human tumor V fragments are cloned and sequenced using afamily-specific primer or primer mixture for leader and constant regionsequences. Next, scFv is constructed using primers based on the sequenceof each V gene cloned. These primers can have specific restrictionendonuclease sites to facilitate routine cloning, or scFv is made byoverlapping PCR, according to methods well known in the art. The vectorexpressing the fusion polypeptide can contain several unique restrictionendonuclease sites (e.g., XhoI, BamHI) between the 3′ end of the spacersequence and the 5′ end of the c-myc and six His tag sequences, or the5′ end of the polyA transcription terminator region (if a SmaI site isused), thus enabling routine cloning of any scFv, tumor antigen or viralantigen.

As described herein, nucleic acid encoding the viral chemokine-tumorantigen fusion polypeptides of this invention is expressed in yeast(e.g., Saccharomyces cerevisiae; Pichia pastoris, etc.) or in mammaliancell culture according to methods standard in the art. The proteinsproduced in these systems are affinity purified with anti-c-mycantibodies (e.g., 9E10; M5546, Sigma) or anti-poly-His antibodies (e.g.,H1029, Sigma). Alternatively, immobilized metal chelate affinitychromatography (Ni-NTA resin, Qiagen) is used for purification ofsoluble or refolded fusion polypeptides.

Administration of fusion polypeptides to human subjects. Immunity andsuppression of tumor growth in a human subject. To elicit a tumor cellgrowth-inhibiting response in a human subject, a fusion polypeptidecomprising a viral chemokine and a tumor antigen which is present in thehuman subject is administered to the subject subcutaneously in a doseranging from 1 to 500 μg of the fusion polypeptide once weekly for abouteight weeks or once monthly for about six months. Within the first monthfollowing the initial immunization, blood samples can be taken from thesubject and analyzed to determine the effects of administration of thefusion polypeptide. Particularly, the presence in the subject's serum,of antibodies reactive with the tumor antigen in the fusion protein canbe determined by ELISA, Western blotting or radioimmunoprecipitation, orother methods for detecting the formation of antigen/antibody complexesas would be standard practice for one of ordinary skill in the art ofimmunology. Also, a cellular immune response to the tumor antigen in thefusion polypeptide can be detected by peripheral blood lymphocyte (PBL)proliferation assays, PBL cytotoxicity assays, cytokine measurements, orother methods for detecting delayed type hypersensitivity and cellularimmune response, as would be standard practice for one of ordinary skillin the art of immunology. Additionally, the kinetics of tumor growth andinhibition of tumor cell growth can be determined by monitoring thesubject's clinical response, through physical examination, tumormeasurement, x-ray analysis and biopsy. The exact dosage can bedetermined for a given subject by following the teachings as set forthherein, as would be standard practice for one of ordinary skill in theart of vaccine development.

Example 3

This example demonstrates that antigens elicit effective immunity whenthey are targeted to APC, particularly iDC, via chemokine receptors asfusions with proinflammatory chemokines factors. Moreover, this exampledemonstrates that proinflammatory factors such as murine β-defensinsinduce chemotaxis of immature, but not mature, DC and, thus, can serveeffectively as an carrier for targeting antigens to APC. Non-immunogenictumor antigens or xenogeneic HIV gp120 antigen were rendered effectivelyimmunogenic when immunized as fusion with murine β-defensin 2. Thisexample also demonstrates the use of xenogeneic human and viralchemokines such as Kaposi's sarcoma virus derived chemokine analogues ofhuman MIP-1s, designated vMIP1 and vMIP2 (Nicholas, J., V. R. Ruvolo, W.H. Burns, G. Sandford, X. Wan, D. Ciufo, S. B. Hendrickson, H. G. Guo,G. S. Hayward, and M. S. Reitz. 1997. Kaposi's sarcoma-associated humanherpesvirus-8 encodes homologues of macrophage inflammatory protein-1and interleukin-6. Nat. Med. 3:(3)287-292), which bind multiplereceptors on murine cells and which might circumvent the potentialdevelopment of autoimmunity against host chemokine carriers (Luster, A.D. 1998. Chemokines-chemotactic cytokines that mediate inflammation. N.Engl. J. Med. 338:(7)436-445; Pelchen-Matthews, A., N. Signoret, P. J.Klasse, A. Fraile-Ramos, and M. Marsh. 1999. Chemokine receptortrafficking and viral replication. Immunol Rev. 168:33-49:33-49). Inaddition, using a pair of viral chemokines that bind to CCR8, theagonist vMIP1 (Endres, M. J., C. G. Garlisi, H. Xiao, L. Shan, and J. A.Hedrick. 1999. The Kaposi's sarcoma-related herpesvirus (KSHV)-encodedchemokine vMIP-I is a specific agonist for the CC chemokine receptor(CCR)8. J. Exp. Med. 189:(12)1993-1998) and antagonist MC148, expressedby Molluscum contagiosum virus (MCV) (Luttichau, H. R., J. Stine, T. P.Boesen, A. H. Johnsen, D. Chantry, J. Gerstoft, and T. W. Schwartz.2000. A highly selective CC chemokine receptor (CCR)8 antagonist encodedby the poxvirus molluscum contagiosum. J. Exp. Med. 191:(1)171-180),this example demonstrates that antigen targeting alone in the absence ofchemotaxis is sufficient for induction of immunity.

Methods

Fusion gene cloning and plasmid constructions. Cloning strategy forlymphoma specific V_(H) and V_(L) fragments from 38C13 (Bergmanm Y. andJ. Haimovich. 1977. Characterization of a carcinogen-induced murine Blymphocyte cell line of C3H/eb origin. J. Immunol. 7:413-417) and A20(Kim, K. J., L. C. Kanellopoulos, R. M. Merwin, D. H. Sachs, and R.Asofsky. 1979. Establishment and characterization of BALB/c lymphomalines with B cell properties. J. Immunol. 122:(2)549-554) cells as sFv38and sFv20, respectively, has been reported elsewhere (Biragyn, A., K.Tani, M. C. Grimm, S. D. Weeks, and L. W. Kwak. 1999. Genetic fusion ofchemokines to a self tumor antigen induces protective, T-cell dependentantitumor immunity. Nature Biotechnology 17:253-258). Genes for maturehuman and murine chemokines and defensins were cloned in frame to the5′-end of sFv by RT/PCR from total RNA using specific primers asdescribed previously (Biragyn, A., K. Tani, M. C. Grimm, S. D. Weeks,and L. W. Kwak. 1999. Genetic fusion of chemokines to a self tumorantigen induces protective, T-cell dependent antitumor immunity. NatureBiotechnology 17:253-258). For example: genes for human MDC (GeneBank#HSU83171) and SDF-1β(GeneBank #HSU16752) were cloned from LPS 10 ng/mltreated human monocytes using respectively pairs of primers PRhMDC-5′(CTCTAGACACCATGGCTCGCCTACAGACTGCACT; SEQ ID NO:16) and PRhMDC-3′(TGAATTCTTGGCTCAGCTTATTGAGAATCA; SEQ ID NO:17); and PRhSDF1β-5′(CTCTAGACACCATGAACGCCAAGGTCGTGGTCGTGCTG; SEQ ID NO:18) and PRhSDF1β-3′(TGAATTCCATCTTGAACCTCTTGTTTAAAGCTTT; SEQ ID NO:19). Murine (3-defensin 2(GeneBank #AJ011800) and β-defensin 3 (GeneBank #AF092929) genes werecloned from LPS (10 ng) Balb/c mouse skin using pairs of primers,respectively PRmDF2β-5′ (ACCATGGAACTTGACCACTGCCACACC; SEQ ID NO:20) andPRmDF2β-3′ (TGAATTCAAGATCTTTCATGTACTTGCAACAGGGGTTGTT; SEQ ID NO:21) andPRmDF3β-5′ (ACCATGGAAAAAATCAACAATCAGTAAGTTGTTTGAGG; SEQ ID NO:22) andPRmDF3β-3′ (CTCGAGCTAGAATTCTTTTCTCTTGCAGCATTTGAGGAAA; SEQ ID NO:23).Similarly, murine β-pro-defensin 2 gene was cloned for eukaryoticexpression using PrproDF2βL-5′ (AAAGCTTCCACCATGAGGACTCTCTGCTCT; SEQ IDNO:24) and PRmDF2β-3′, which contained native secretion signal sequence.For bacterial expression, the signal sequence was removed usingPRmDF2β-5′ (ACCATGGCTGTTGGAAGTTTAAAAAGTATTGGA; SEQ ID NO:25) andPRmDF2β-3′. Viral chemokine genes vMIP-I (GeneBank #KSU74585) andvMIP-II (GeneBank #KSU67775) were cloned from BCBL-1 lymphoma cell lineinfected with HHV-8 (NIH AIDS Research & Ref. Reag. Program), usingpairs of primers, respectively PRvMIP1L-5′(TAAGCTTCCACCATGGCCCCCGTCCACGTTTTATGCT; SEQ ID NO:26) and PRvMIP1-3′(TGAATTCAGCTATGGCAGGCAGCCGCTGCATCAGCTGCCT; SEQ ID NO:27) andPRvMIP1IL-5′ (TAAGCTTCACCATGGACACCAAGGGCATCCTGCTCGT; SEQ ID NO:28) andPRvMIP1I-3′ (TGAATTCGCGAGCAGTGACTGGTAATTGCTGCAT; SEQ ID NO:29). Maturesequences of vMIP-I and vMIP-II for expression in bacterial system werecloned using the following pairs of primers: PRvMIP1M-5′(ACCATGGCGGGGTCACTCGTGTCGTACA; SEQ ID NO:30) and PRvMIP1-3′ andPRvMIP2M-5′ (ACCATGGGAGCGTCCTGGCATAGA; SEQ ID NO:31) and PRvMIP1I-3′.MC148 chemokine gene (GeneBank #U96749) was cloned from plasmid DNAcontaining a portion of Moluscum contagiosum virus type 1 genome (Damon,I., P. M. Murphy, and B. Moss. 1998. Broad spectrum chemokineantagonistic activity of a human poxvirus chemokine homolog. Proc. Natl.Acad. Sci. U.S.A 95:(11)6403-6407) using pairs of primers PRMC148L-5′(AAAGCTAGCACCATGAGGGGCGGAGACGTCTTC; SEQ ID NO:32) and PRMC148-3′(AGAATTCCAGAGACTCGCACCCGGACCATAT; SEQ ID NO:33) and PRMC148M-5′(ACCATGGCACTCGCGAGACGGAAATGTTGTTTGAAT; SEQ ID NO:34) and PRMC148-3′,respectively for eukaryotic and bacterial expression. Thecarboxy-terminus of sFv was fused in frame with a tag sequence codingc-myc peptide AEEQKLISEEDLA (SEQ ID NO:35) and six His, respectively.Chemokine and defensins have been fused with sFv through a spacersequence NDAQAPKS (SEQ ID NO:36). To generate constructs encoding mutantchemokines, the first Cys residue was replaced to Ser for allchemokines, except for hMCP-3 and hMDC where the amino-terminus up tothe second Cys residue were truncated. Bacterial expression vectorscontained only genes encoding mature peptide genes, while constructs forDNA vaccination were fused in frame to a leader sequence of IP-10 inpCMVE/AB, except for constructs designed for pMDCsFv38 and pSDF1βsFv38plasmids, which contained their native signal sequences. All constructswere verified by DNA dideoxy-sequencing method, using T7 sequenase kit(Amersham, USA) and purified using Qiagen plasmid purification kit(Qiagen, Valencia, Calif.).

For the second model, gp120 gene was cloned from the plasmid DNAcontaining portion of HIV-1 (isolate 89.6) in frame with IP-10 secretionsignal sequence (pgp120) using primers PRM89.6ENV-5′(AAAGTCGACAAAGAAAAAACGTGG GTCACAATCT; SEQ ID NO:37) and PR89.6ENV-3′(ATTCCCGGGTTATTTTTCTCTTTGCACTGTTCTTCTC; SEQ ID NO:38). Similarly, gp120was fused in frame with coding sequences from murine β-defensin 2, hMDCand hMCP3 to generate DNA constructs pmDF2βgp120, phMDCgp120,phMCP3gp120, respectively.

Recombinant fusion proteins purified as inclusion bodies after 8 hoursof induction in Super-Broth (Digene Diagnostics, Inc., Beltsville, Md.)with 0.8 mM IPTG in the presence of 150 μg/ml carbenicillin and 50 μg/mlampicillin at 30° C., and refolded according to Buchner, et al (Buchner,J., I. Pastan, and U. Brinkmann. 1992. A method for increasing the yieldof properly folded recombinant fusion proteins: single-chainimmunotoxins from renaturation of bacterial inclusion bodies. Anal.Biochem. 205:(2)263-270) with modifications (Biragyn, A., K. Tani, M. C.Grimm, S. D. Weeks, and L. W. Kwak. 1999. Genetic fusion of chemokinesto a self tumor antigen induces protective, T-cell dependent antitumorimmunity. Nature Biotechnology 17:253-258) from BL21(DE3) cells(Invitrogen, Milford, Mass.). The refolded fusion proteins were purifiedby heparin-sepharose chromatography (Pharmacia Biotech, Uppsala,Sweden). The integrity and purity of recombinant proteins were tested byPAGE under reducing conditions and by western blot hybridization with9E10 anti-c-myc mAb (Sigma). Purification usually yielded solubleprotein with greater than 90% purity. Correct folding of purified sFv38proteins were determined by the ability to bind to anti-idiotype mAbS1C5 by ELISA (Biragyn, A., K. Tani, M. C. Grimm, S. D. Weeks, and L. W.Kwak. 1999. Genetic fusion of chemokines to a self tumor antigen inducesprotective, T-cell dependent antitumor immunity. Nature Biotechnology17:253-258). Briefly, serially diluted sFv were added to microtiterplates coated with 10 μg/ml anti-c-myc mAb 9E10. After washing, plateswere incubated with a 1:300 diluting of biotinylated S1C5. Plates werewashed, incubated with streptavidin-HRP (1:5000, Jackson Immuno researchLab., Inc., Bar harbor, Me.) and developed with ABTS peroxidasesubstrate (KPL, Gaithersburg, Md.).

Isolation of murine bone marrow derived dendritic cells. Murine bonemarrow derived DC were isolated as described elsewhere (Fields, R. C.,J. J. Osterholzer, J. A. Fuller, E. K. Thomas, P. J. Geraghty, and J. J.Mule. 1998. Comparative analysis of murine dendritic cells derived fromspleen and bone marrow. J. Immunother. 21:(5)323-339). Briefly, bonemarrow was collected from tibias and femurs of 4 to 6 months old BALB/cmice by flushing with PBS using a 10 mL syringe with a 27 gauge needle.Erytrocytes were lysed by treatment with ACK lysis buffer (BioWhittaker,Walkersville, Md.). Cells expressing CD8, CD4, B220 and I-A^(b) weredepleted using a mixture of mAbs and rabbit complement. The mAbs wereTIB-146 (anti-B220), TIB-150 (anti-CD8), TIB-207 (anti-CD4), TIB-229(anti-1-A^(b)) obtained from ATCC. Cells were then resuspended with DCmedium (RPMI 1640 containing 5% heat inactivated fetal bovine serum, 1%penicillin streptomycin, 1% L-glutamine and 5×10⁻⁵ 2-ME) supplementedwith 10 ng/mL recombinant murine IL-4 and 10 ng/mL recombinant murineGM-CSF (Peprotech) and cultured in 6 well plates (7×10⁵ cells/mL, 5mL/well). On day two, 200 μl DC medium containing 10 ng/mL of both IL-4and GM-CSF were added to each well. Then, at day four, 5 μl of DC mediumcontaining cytokines were added to each well after non-adherent cellswere removed, and cells were cultured for additional 4 days. Cells wereharvested on day 4 and day 7 and used in subsequent experiments.

In vitro chemotaxis assay: The chemotactic migration of murine dendriticcells was assessed using a 48-well microchemotaxis chamber technique aspreviously described (Falk, W., R. H. J. Goodwin, and E. J. Leonard.1980. A 48-well micro chemotaxis assembly for rapid and accuratemeasurement of leukocyte migration. J. Immunol Methods 33:(3)239-247;Yang, D., Q. Chen, S. Stoll, X. Chen, O. M. Howard, and J. J. Oppenheim.2000. Differential regulation of responsiveness to fMLP and C5a upondendritic cell maturation: correlation with receptor expression.165:(5)2694-2702). Briefly, different concentrations of 26 mlchemotactic factors or aliquots of sFv fusion protein, serially dilutedin chemotaxis medium (RPMI 1640, 1% BSA, 25 mM HEPES), were placed inthe lower compartment of the chamber (Neuro Probe, Cabin John, Mass.),and 50 μl of dendritic cells (10⁶ cells/ml) were added to wells of theupper compartment. The lower and upper compartments were separated by a5-μm polycarbonate filter (Osmonics, Livermore, Calif.). Afterincubation at 37° C. in humidified air with 5% CO₂ for 1.5 h, thefilters were removed, scraped, and stained. Dendritic cells migratingacross the filter were counted with the use of a Bioquant semiautomaticcounting system. The results are presented as chemotactic index (C.I.)defined as the fold increase in the number of migrating cells in thepresence of test factors over the spontaneous cell migration (in theabsence of test factors). The results are expressed as the mean ±SE oftriplicate samples. MIP-3α and MIP-3β were purchased from PeproTech(Rocky Hill, N.J.).

Chemokine receptor binding assay. Binding assays were performed by usinga single concentration of radio-labeled MIP-1β or SDF-1α (human[¹²⁵-I]-[Leu³, Gly⁴⁷]-MIP-1β and human [¹²⁵I]-SDF-1α, 2200 Ci/mmol, NENLife Science Products Inc., Boston, Mass.) in the presence of increasingconcentrations of unlabeled ligands (MIP-1β and SDF-1α obtained fromPeproTech, Rocky Hill, N.J.). Human HEK293 cells transfected with CCR5at 1×10⁶/sample were suspended in 200 μl binding medium composed ofRPMI1640, 1 mg/ml BSA, 25 mM HEPES, and 0.05% sodium azide, andincubated in duplicates at room temperature for 40 min. Afterincubation, the cells were pelleted through a 10% sucrose/PBS cushionand the radioactivity associated with cell pellets was determined in aγ-counter (Clinigamma-Pharmacia, Gaithersburg, Md.). The binding datawere then analyzed with a Macintosh computer program LIGAND (P. Munson,Division of Computer Research and Technology, NIH, Bethesda, Md.). Thedegree of competition for binding by unlabeled chemokines was calculatedas follows: % competition for binding=1−(cpm obtained in the presence ofunlabeled ligand/cpm obtained in the absence of unlabeled ligand)×100%.

HIV-1 env antibody and CTL assays. Five BALE/c female mice per groupwere gene-gun immunized with DNA plasmids four times using gene-gun. Twoweeks after the last immunization, HIV-1 89.6 env specific CTL wasassessed in spleens and Peyer's patches as described elsewhere(Belyakov, I. M., M. A. Derby, J. D. Ahlers, B. L. Kelsall, P. Earl, B.Moss, W. Strober, and J. A. Berzofsky. 1998. Mucosal immunization withHIV-1 peptide vaccine induces mucosal and systemic cytotoxic Tlymphocytes and protective immunity in mice against intrarectalrecombinant HIV-vaccinia challenge. Proc. Natl. Acad. Sci. U.S.A95:(4)1709-1714). Briefly, immune cells from spleen or Peyer's patchwere cultured at 5×10⁶ per/milliliter in 24-well culture plates incomplete T cell medium (CTM): RPMI 1640 containing 10% fetal bovineserum, 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (100mg/ml), and 5×10′ M 2-mercaptoethanol. Three days later 10% concanavalinA supernatant was added as a source of IL-2 (T-STIM, CollaborativeBiomedical Products, Bedford, Mass.). Spleen or Peyer's patch cells werestimulated in vitro with P18-89.6A9 peptide (IGPGRAFYA; SEQ ID NO:39)(Belyakov, I. M., L. S. Wyatt, J. D. Ahlers, P. Earl, C. D. Pendleton,B. L. Kelsall, W. Strober, B. Moss, and J. A. Berzofsky. 1998. Inductionof a mucosal cytotoxic T-lymphocyte response by intrarectal immunizationwith a replication-deficient recombinant vaccinia virus expressing humanimmunodeficiency virus 89.6 envelope protein. J. Virol.72:(10)8264-8272) for a 7-day culture periods before assay. Cytolyticactivity of CTL lines was measured by a 4-hour assay with ⁵¹Cr-labeledP815 cell targets. For testing the peptide specificity of CTL,⁵¹Cr-labeled P815 targets were pulsed for 2 hours with peptide at thebeginning of the assay or left unpulsed as controls. The percentspecific ⁵¹Cr release was calculated as 100×(experimentalrelease−spontaneous release)/(maximum release−spontaneous release).Maximum release was determined from supernatants of cells that werelysed by addition of 5% Triton-X 100. Spontaneous release was determinedfrom target cells incubated without added effector cells (Belyakov, I.M., M. A. Derby, J. D. Ahlers, B. L. Kelsall, P. Earl, B. Moss, W.Strober, and J. A. Berzofsky. 1998. Mucosal immunization with HIV-1peptide vaccine induces mucosal and systemic cytotoxic T lymphocytes andprotective immunity in mice against intrarectal recombinant HIV-vacciniachallenge. Proc. Natl. Acad. Sci. U.S.A 95:(4)1709-1714).

Serum anti-env antibodies assessed by ELISA on 5 μg/ml gp120 proteinfrom isolate 89.6 produced in vaccinia virus coated 96-well plate. Thebound antibodies were detected by goat anti-mouse Ig-HRP mAb (Caltag)and developed with ABTS peroxidase substrate (KPL, Gaithersburg, Md.).

Tumor cell lines and mice. The carcinogen-induced, C3H 38C-13 B celllymphoma (Bergmanm Y. and J. Haimovich. 1977. Characterization of acarcinogen-induced murine B lymphocyte cell line of C3H/eb origin. J.Immunol. 7:413-417) was obtained from R. Levy (Stanford, Calif.). The38C-13 tumor secretes and expresses IgM (k) on the cell surface.Inoculation of as few as 10² 38C-13 tumor cells i.p. into normalsyngeneic mice results in progressive tumor growth and death of the hostwith a median survival time of only two weeks. Mice surviving past 60days from tumor challenge are long-term survivors. The BALB/c A20lymphoma (Kim, K. J., L. C. Kanellopoulos, R. M. Merwin, D. H. Sachs,and R. Asofsky. 1979. Establishment and characterization of BALB/clymphoma lines with B cell properties. J. Immunol. 122:(2)549-554) wasobtained from the American Type Culture Collection (Rockville, Md.) andexpresses IgGk. 38C-13 and A20 cells from a common frozen stock werepassaged in vitro 3 days before use in RPMI 1640 supplemented with 100U/ml of penicillin and streptomycin, 2×10⁻⁵M 2-mercaptoethanol, and heatinactivated 10% fetal bovine serum (Gibco BRL, Gaithersburg, Md.).

In vivo immunizations and tumor protection experiment. Animal care wasprovided in accordance with the procedures outlined in a Guide for theCare and Use of Laboratory Animals (NIH Publication No. 86-23, 1985).Six- to nine-week old female C3H/HeNCrlBR or BALB/c mice (Charles RiverLaboratories, Frederick, Md.) were used. Syngeneic C3H/HeN or BalbC mice(10 per group) were immunized with Hellos Gene Gun System (Bio-Rad,Hercules, Calif.) with plasmid DNA three times every two weeks. Theabdominal area of mice was shaved, and 1μ gold particles (Bio-Rad,Hercules, Calif.) carrying 1-3 μg DNA were injected at 400 psi. Twoweeks after the last immunization, mice were challenged i.p. with 200038C-13 lymphoma cells from a single preparation of tumor and followedfor survival. Differences in survival between groups were determined bynon-parametric logrank test (BMDP statistical software, Los Angeles).P-values refer to comparison with group immunized with DNA expressingthe same chemokine or defensin fused with an irrelevant sFv, or sFvfused with mutant chemokine, unless specified.

Therapy of established tumor with DNA vaccine. Six- to nine-week oldfemale BALB/c mice (ten per group) were challenged with 2.5×10⁵syngeneic A20 tumor cells. At day 1, 4, 8 and 18 these mice weregene-gun immunized with DNA plasmid (containing about 1-2 μg DNA perimmunization) and mice followed for tumor progression.

Results

Property of constructs: murine β-defensins and viral chemokines retaintheir functional integrity when produced as fusion proteins with sFv.First, a variety of chemokine and β-defensin fusion proteins with sFv, alymphoma Ig-derived non-immunogenic Fv, were cloned and purified (Table1). The functional integrity of these proteins were tested by theability to induce chemotaxis of murine APC and THP-1 cells. As expected,chemokine fusion proteins induced dose-dependent chemotaxis. THP-1 cellswere chemo-attracted to vMIP1, human MCP-3, and SDF-10 fusion proteins,but not to the fusion with antagonist chemokine vMIP2, vMIP2sFv38.Control mutant fusion proteins generated for the each chemokine byreplacing the first Cys residue by Ser or by truncation of theamino-termini, as expected, did not induce chemotaxis of THP-1 cells ormurine DC. Furthermore, vMIP2 fusion proteins were tested for theirability to bind to their respective receptor (-s). vMIP2sFv38 coulddisplace labeled MIP1β and SDF1α in a dose dependent manner from CCR5and CXCR4 transfected cell lines, respectively. In contrast, nodisplacement was detected by vMIP2MsFv38 fusion protein, which containeda replacement Cys/Ser mutation in vMIP2 or by control sFv protein alone.

Since human β-defensin 2 was reported to act via CCR6, murine β-defensinfusion proteins were assayed for their ability to induce chemotaxis ofdifferent subsets of murine cells. β-defensin fusion proteins inducedchemotaxis of murine bone marrow derived iDC in a dose dependent mannerwith peak activity at 10 ng/ml and 100 ng/ml for Def2βsFv38 andDef3βsFv38, respectively. The immature phenotype of these DC (seeMethods) was also supported by their ability to migrate to human MIP3α,a chemo-attractant specific for CCR6⁺ immature DC (Dieu, M. C., B.Vanbervliet, A. Vicari, J. M. Bridon, E. Oldham, S. Ait-Yahia, F.Briere, A. Zlotnik, S. Lebecque, and C. Caux. 1998. Selectiverecruitment of immature and mature dendritic cells by distinctchemokines expressed in different anatomic sites. J. Exp. Med188:(2)373-386; Yang,

TABLE 1 Ligand-Antigen fusion constructs DNA vaccine Ligand: Defensinname or chemokine Antigen Protein Name Description Antigen alone psFv38none sFv38 sFv38 Single chain antibody fragment from 38C-13 lymphomapsFv20 none sFv20 sFv20 Single chain antibody fragment from A20 lymphomapgp120 none gp120 gp120 gp120 antigen (HIV-1, isolate 89.6) Defensinfusions: pmDF2βsFv38 murine β-defensin 2 sFv38 mDF2βsFv38 Murineβ-defensin 2 fusion with sFv38 pmDF2βgp120 murine β-defensin 2 gp120mDF2βgp120 Murine β-defensin 2 fusion with gp120 (HIV-1, 89.6)pmDF3βsFv38 murine β-defensin 3 sFv38 mDF3βsFv38 Murine β-defensin 3fusion with sFv38 pproDF2βsFv38 murine pro-β-defensin 2 sFv38mproDF2βsFv38 Murine pro-β-defensin 2 fusion with sFv38 Viral chemokineFusions: pvMIP2sFv38 viral MIP2 sFv38 vMIP2sFv38 Viral MIP2 fusion withsFv38 pvMIP1sFv38 viral MIP1 sFv38 vMIP1sFv38 Viral MIP1 fusion withsFv38 pMC148sFv38 MC148 sFv38 MC148sFv38 Viral MC148 fusion with sFv38Pro-inflammatory Chemokine fusions: phMCP3sFv38 Human MCP-3 sFv38hMCP3sFv38 Human MCP-3 fusion with sFv38 phMCP3sFv20 Human MCP-3 sFv20hMCP3sFv20 Human MCP-3 fusion with sFv20 phMCP3gp120 Human MCP-3 gp120hMCP3gp120 Human MCP-3 fusion with gp120 (HIV-1, isolate 89.6)phMDCsFv38 Human MDC sFv38 hMDCsFv38 Human MDC fusion with sFv38phMDCsFv20 Human MDC sFv20 hMDCsFv20 Human MDC fusion with sFv20phMDCgp120 Human MDC gp120 hMDCgp120 Human MDC fusion with gp120 (HIV-1,isolate 89.6) phSDF1βsFv38 Human SDF-1β sFv38 hSDF1βsFv38 Human SDF1-βfusion with sFv38 Mutant chemokine fusions: pvMIP2MsFv38 Mutant vMIP2sFv38 vMIP2MsFv38 Mutant vMIP2 fusion with sFv38 pvMIP1MsFv38 MutantvMIP1 sFv38 vMIP1MsFv38 Mutant vMIP1 fusion with sFv38 phMCP3MsFv38Mutant hMCP-3 sFv38 hMCP3MsFv38 Mutant human MCP-3 fusion with sFv38phMDCMsFv38 Mutant hMDC sFv38 hMDCMsFv38 Mutant human MDC fusion withsFv38 Control fusions: pmDF3βMuc1 β-defensin 3 Muc-1 mDF3βMuc1 Murineβ-defensin 3 fusion with 80 aa hMuc-1 peptide phMCP3-EGFP Human MCP-3EGFP hMCP3-EGFP Human MCP-3 fusion with EGFP protein phMDC-EGFP HumanMDC EGFP hMDC-EGFP Huma MDC fusion with EGFP protein Protein vaccine:Ig38-KLH None Ig38 Ig38-KLH 38C-13 lymphoma derived IgM proteincross-linked with KLH Ig20-KLH none Ig20 Ig20-KLH A20 lymphoma derivedIgG2a protein cross-linked with KLHD., O. M. Howard, Q. Chen, and J. J. Oppenheim. 1999. Cutting edge:immature dendritic cells generated from monocytes in the presence ofTGF-beta 1 express functional C-C chemokine receptor 6. J. Immunol163:(4)1737-1741), and their inability to react to human MIP3β(Sallusto, F., B. Palermo, D. Lenig, M. Miettinen, S. Matikainen, I.Julkunen, R. Forster, R. Burgstahler, M. Lipp, and A. Lanzavecchia.1999. Distinct patterns and kinetics of chemokine production regulatedendritic cell function. Eur. J. Immunol 29:(5)1617-1625), achemo-attractant specific for CCR7⁺ mature DC. None of the defensinfusion proteins stimulated chemotaxis of mature DC, which migrated toMIP3β. Control fusion protein proDef2βsFv38 did not induce chemotaxis ofany DC. Therefore, murine β-defensin 2 and 3 fusion proteins canspecifically target immature DC. Furthermore, sFv fusion with β-defensinand xenogeneic human or viral chemokines does not disrupt theirfunctional chemokine properties.

Murine β-defensin, xenogeneic proinflammatory and viral chemokine fusionconstructs render non-immunogenic tumor antigen immunogenic. Next, thesefusion proteins were used to induce specific immunity againstnon-immunogenic sFv38 when administered as a DNA vaccine in mice. Tenmice per group were immunized by gene-gun with plasmids encoding fusionproteins with mature β-defensins, pDef2fβsFv38 and pDef3fβsFv38,respectively, or with human or viral chemokines, pSDF1βsFv38, pMDCsFv38,pvMIP1sFv38 or pvMIP2sFv38. Control mice were immunized with DNAconstructs encoding sFv fused with inactive pro-Defensin(pproDef2βsFv38), or mutated chemokines (phMCP3MsFv38, pMDCMsFv38,pMIP1MsFv38 and pvMIP2MsFv38). Mice immunized with plasmids encoding sFvfusion proteins with both murine β-defensins, murine MCP-3, human MDC orviral chemokines induced significant idiotype-specific antibodies whichwere comparable to the levels induced by vaccination with tumor-derivedintact Ig protein conjugated to KLH. In contrast, control mice immunizedwith an inactive pro-β-defensin (pproDef2βsFv38) or mutant chemokine sFvfusion constructs (hpMCP3MsFv38, pMDCMsFv38 and pvMIP2MsFv38), or sFv38alone did not produce any anti-Id antibody responses. Moreover, amixture of separate plasmids encoding (β-defensin 3 (pmDF3βMuc1T) andsFv antigen (sFv38) failed to elicit a specific humoral response,demonstrating a requirement for sFv to be physically linked toβ-defensin or chemokine moiety. Therefore, non-immunogenic sFv wasrendered immunogenic by fusion with mature murine β-defensins, syngeneicmurine, xenogeneic human or viral chemokines. These results also suggestthat it was not sufficient to simply attract APC to the site ofproduction of sFv, but that direct APC targeting with self-antigen fusedto β-defensin or chemokine was required. Thus, induction of anti-Idantibodies by sFv fusion proteins appeared to involve receptor-mediatedbinding and delivery of antigen to APC. Vaccination elicited anti-Idantibodies of various isotypes, suggesting activation of differenteffector cells by different pro-inflammatory moieties. All carriersinduced specific IgG1 antibodies. However, MDC containing vaccines alsoelicited high titers of IgG2b and IgG3, while fusion constructs withβ-defensin-3 and vMIP2 elicited high titers of IgG2b but little IgG3,and little IgG2b or IgG3, respectively.

Two weeks after the last of three serial immunizations, mice werechallenged with a 20-fold lethal dose of syngeneic tumor. No survivalwas observed in control groups immunized with PBS or plasmids encodingsFv38 fused with inactive pro-β-defensin-2 (pproDef2βsFv38), MDC alone(pMDC-EGFP), or with mutant constructs pMDCMsFv38, pvMIP2MsFv38,pvMIP1MsFv38. Moreover, no protection was observed in mice immunizedwith DNA encoding a mixture of unlinked functionally active β-defensinor chemokine with sFv. In contrast, significant protective immunity waselicited in mice immunized with pDef2βsFv38 and pDef3βsFv38 (logrankP<0.001 as compared with pproDef2βsFv38 and pMIP2MsFv38, respectively).Similarly, pMDCsFv38, pvMIP2sFv38 and pvMIP1sFv38 immunized micedemonstrated statistically significantly prolonged survival (logrankP<0.001 as compared with pMDC-EGFP and mutant pvMIP2MsFv38 andpvMIP1MsFv38, respectively). Therefore, β-defensins and xenogeneic humanand viral chemokines can render a non-immunogenic self-tumor antigen(sFv) immunogenic and elicit specific antitumor immunity. These dataalso suggest that chemokine receptor engagement with chemokine- ordefensin-sFv fusion is useful for the induction of immunity.

Requirement for secretion but not for chemotaxis for induction of immuneresponses. Additional constructs were used for testing in vivo. Micewere immunized with MCP-3 fusion constructs with or without a secretoryleader sequence (pMCP3sFv38 and pMCP3sFv38-w/o-SL, respectively). Highlevels of Id-specific antibodies were detected in mice immunized withpMCP3sFv38, containing an intact secretory leader; however, noantibodies were elicited in mice immunized with pMCP3sFv38-w/o-SL.Furthermore, tumor protection was elicited only in those mice immunizedwith pMCP3sFv38, but not with pMCP3sFv38-w/o-SL (logrank P<0.001). Inaddition, no protection was detected in mice immunized with DNAexpressing a secretable but mutated MCP-3 fusion protein, which couldnot bind the respective receptor (pMCP3MsFv38). These data areconsistent with immunity being induced by APC which took up (viachemokine receptor) a functionally active chemokine fusion protein whichwas secreted from bystander cells, rather than by APC directly beingtransduced by gene gun immunization.

It was determined whether activation of receptor-mediated chemotaxis wasrequired for eliciting immune responses, or whether receptor bindingalone was sufficient. Immune responses of a selective pair of agonist(vMIP1) (Endres, M. J., C. G. Garlisi, H. Xiao, L. Shan, and J. A.Hedrick. 1999. The Kaposi's sarcoma-related herpesvirus (KSHV)-encodedchemokine vMIP-I is a specific agonist for the CC chemokine receptor(CCR)8. J. Exp. Med. 189:(12)1993-1998) and antagonist (MC148)(Luttichau, H. R., J. Stine, T. P. Boesen, A. H. Johnsen, D. Chantry, J.Gerstoft, and T. W. Schwartz. 2000. A highly selective CC chemokinereceptor (CCR)8 antagonist encoded by the poxvirus molluscumcontagiosum. J. Exp. Med. 191:(1)171-180) chemokines, which bind toCCR8, were compared to immune responses in mice immunized with plasmidsencoding pvMIP1 and MC148 fusion proteins, respectively (pMIP1sFv38 andpMC148sFv38): Mice immunized with either of these chemokine fusionconstructs produced comparable levels of specific antibodies. Moreover,significant tumor protection was detected in both groups of micechallenged with a high dose of 38C-13 cells (logrank P<0.001 forpMC148sFv38 and P<0.01 for pvMIP1sFv38, respectively, compared withcontrol pvMIP1MsFv38). These data suggest that chemokine receptorbinding alone, in the absence of subsequent signaling for chemotaxis, issufficient to induce immunity.

Discussion

This example demonstrates that use of proinflammatory factors of innateand adaptive immunity such as β-defensins 2 and 3 and viral chemokines(Luster, A. D. 1998. Chemokines-chemotactic cytokines that mediateinflammation. N. Engl. J. Med. 338:(7)436-445) can help to elicit strongimmune responses both against a model non-immunogenic tumor antigen,lymphoma idiotype (Stevenson, F. K., D. Zhu, C. A. King, L. J. Ashworth,S. Kumar, and R. E. Hawkins. 1995. Idiotypic DNA vaccines against B-celllymphoma. Immunol. Rev. 145:211-228), and viral antigen, HIV gp120. Theappeal of this approach was based not only on the ability of thesemediators of innate and adaptive immunity to target surface receptors onAPC, particularly on iDC (Yang, D., O. Chertov, S. N. Bykovskaia, Q.Chen, M. J. Buffo, J. Shogan, M. Anderson, J. M., Schroder, J. M. Wang,O. M. Howard, and J. J. Oppenheim. 1999. Beta-defensins: linking innateand adaptive immunity through dendritic and T cell CCR6. Science286:(5439)525-528), presumably resulting in increased uptake of antigen,but possibly also to induce of expression of co-stimulatory moleculesand, in turn, production of other pro-inflammatory cytokines andfactors. This example demonstrates that both murine β-defensin 2 and 3efficiently induced chemotaxis of immature, but not mature, murine bonemarrow derived DC, suggesting that a β-defensin specific receptor(s) isexpressed on immature DC.

Example 4

As an example of how the vaccine of this invention can be administeredto a patient to treat cancer or to treat or prevent HIV infection (withthe additional administration of adjuvants, such as immunostimulatorycytokines, if desired), the following is a complete protocol for aclinical trial describing the administration of Id-KLH and GM-CSF topatients to treat follicular lymphoma. The same study design can beemployed for the administration of the viral chemokine-tumor antigenfusion polypeptide or the viral chemokine-viral antigen fusionpolypeptide of the present invention or nucleic acids encoding thefusion polypeptides of this invention, with appropriate modifications,as would be apparent to one of skill in the art. In particular, studiesto test the efficacy of HIV vaccines are well known in the art and theclinical protocol described herein can be readily modified by one ofskill in the art as appropriate to test the efficacy of the HIV fusionpolypeptide or HIV fusion polypeptide-encoding nucleic acid of thisinvention according to well known protocols for testing HIV vaccines(126,127).

Background and Rationale

Immunoglobulin (Ig) molecules are composed of heavy and light chains,which possess highly specific variable regions at their amino termini.The variable regions of heavy and light chains combine to form theunique antigen recognition site of the Ig protein. These variableregions contain determinants that can themselves be recognized asantigens, or idiotopes. B-cell malignancies are composed of clonalproliferations of cells synthesizing a single antibody molecule withunique variable regions in the heavy and light chains. B-cell lymphomasare neoplasms of mature resting and reactive lymphocytes which generallyexpress synthesized Ig at the cell surface. The idiotypic determinantsof the surface Ig of a B-cell lymphoma can thus serve as atumor-specific marker for the malignant clone.

Studies in experimental animals, as well as in man, have demonstratedthe utility of the Ig idiotype as a tumor-specific antigen for the studyof the biology of B-cell lymphoma in vitro and as a target for passiveimmunotherapy in vivo (1, 2, 3). Furthermore, active immunizationagainst idiotypic determinants on malignant B cells has beendemonstrated to produce resistance to tumor growth in a number ofsyngeneic experimental tumor models, as well as specific anti-tumortherapy against established tumors (4-13). These results, takentogether, provided the rationale for testing autologous tumor-derivedidiotypic surface Ig (Id) as a therapeutic “vaccine” against humanB-cell lymphoma. Furthermore, preclinical studies in subhuman primatesdemonstrated that optimal immunization with human lymphoma-derived Idrequired conjugation of the protein to an immunogenic protein carrier(keyhole limpet hemocyanin; KLH) and emulsification in an adjuvant (14).

Guided by these observations, nine patients with B-cell lymphoma wereimmunized with autologous Id protein (15). These patients received noanti-tumor therapy during the time of the study. They were either incomplete remission or in a state of minimal residual disease followingconventional chemotherapy. In addition, three patients with rapidlyprogressive recurrent lymphoma were enrolled in a separate safety study;all three required reinstitution of chemotherapy shortly afterenrollment, did not complete the immunization series, and were notstudied further. They received intramuscular injections of 0.5 mg of Idconjugated to KLH at 0, 2, 6, 10 and 14 weeks, followed by two boosterinjections at 24 and 28 weeks. Patients in the first trial (fivepatients) received Id-KLH alone for the first three immunizations, thenId-KLH emulsified in a Pluronic polymer-based adjuvant vehicleformulation for all subsequent immunizations. Because noidiotype-specific immune responses were observed prior to the additionof the adjuvant to the program in this first group of patients, patientsin the second trial (four patients) received the entire series ofimmunizations with this adjuvant. All patients were analyzed foridiotype-specific antibody production and peripheral blood mononuclearcell (PBMC) proliferative responses in vitro immediately before eachimmunization and at one to two month intervals following the lastimmunization. The KLH carrier provided a convenient internal control forimmunocompetence of the patients and all patients demonstrated bothhumoral and PBMC proliferative responses to the KLH protein, with theexception of one patient, who demonstrated only the latter. Seven of thenine patients demonstrated either a humoral (n=2) or a cell-mediated(n=4) anti-idiotypic immunological response, or both (n=1).

Anti-idiotypic antibody responses were detected by analysis of pro- andhyper-immune sera in either direct, or competition, ELISA. Theimmunization with autologous Id protein induced significant titers ofanti-idiotypic antibody that either directly bound or inhibited thebinding of a murine anti-idiotype monoclonal antibody (anti Id mAb) toId on the plate. The specificity of the humoral response for the Igidiotype was demonstrated by the lack of significant binding ofhyperimmune serum to a panel of isotype-matched human Igs of unrelatedidiotype, or by the lack of significant inhibition of a panel ofheterologous Id-anti-Id systems, respectively. Peak humoral responseswere obtained after the fifth immunization and persisted for at leastnine months. The anti-idiotypic antibody produced by patient 1 wasaffinity-purified and shown to contain heterogeneous light chains aswell as immunoglobulin G heavy chains. This patient's antibody titer wassuccessfully boosted with a single administration of Id-KLH in adjuvantafter a decline of the humoral response after 15 months.

Cellular immune responses were measured by the proliferation of PBMC toKLH and to autologous Id separately at concentrations ranging from 1-100μg per milliliter of soluble protein in five day in vitro cultures. Noneof the pre-immune PBMC demonstrated any preexisting proliferation toautologous Id above that to culture medium alone. Hyperimmune PBMC fromall patients demonstrated strong proliferative responses to the KLHcarrier. Of primary interest, significant hyperimmune proliferativeresponses to Id were detected in five patients. Although their responseswere of lower magnitude than parallel responses to KLH, patients 3, 4,6, 8 and 9 were classified as responders on the basis of reproducibleincreases in counts-per minute (cpm) ³H-thymidine incorporation in wellscontaining Id, compared with medium alone, that were sustained overmultiple time points. Patients demonstrating occasional increases in cpmin wells containing Id compared with medium alone were classified asnon-responders (patients 1 and 5).

Flow cytometry analysis of cultures demonstrating proliferation to Idrevealed a predominance of cells staining positively for CD4 (>95%),suggesting the phenotype of the responding cell subpopulation. Thesecultures could be successfully expanded for approximately four weeks bystimulation alternatively with interleukin-2 (IL-2) and Id-pulsedautologous irradiated PBMC as antigen-presenting cells. Specificity ofthe responses for Ig idiotype was confirmed by the lack of significantproliferation to an isotype-matched human Ig of unrelated idiotypecompared with medium alone. Such idiotype-specific PBMC proliferativeresponses were observed only after the addition of the adjuvant to theprogram and also persisted for at least 9-14 months.

The ability of the idiotype-specific humoral response to bind autologoustumor cells was also tested. This was shown by the inhibition of bindingof a labeled murine anti-idiotype mAb to tumor cells from apre-treatment lymph-node specimen from patient 8 by hyperimmune, but notby pre-immune, serum from this patient. In addition, affinity purifiedanti-idiotypic antibodies from the hyperimmune sera of the two otherpatients who demonstrated idiotype specific humoral responses weredemonstrated by flow cytometry to bind autologous tumor.

All patients were also closely monitored for disease activity withphysical examinations and routine laboratory and radiographic studies.Of the two patients with measurable tumor at the initiation of Idimmunization, one (patient 1) experienced complete regression of asingle 2.5 cm left submandibular lymph node, and the other (patient 4)experienced complete regression of a 4.5 cm cutaneous lymphomatous masson the right arm. This clinical response in patient 4 correlated with anId-specific, PBMC proliferative response in vivo. Correlating with theduration of their immunological responses, the clinical responses inboth patients have continued at 24 and 10 months, respectively, aftercompletion of the immunization series. Moreover, with a median follow uptime of 10 months, the only case of tumor recurrence among thosepatients who were in remission and completed the immunization seriesoccurred in patient 5, who was one of the two patients who failed todemonstrate an idiotype-specific immunological response.

Toxicity was minimal in all twelve patients. All patients experiencedtransient local reactions characterized by mild erythema, induration,and discomfort, without skin breakdown, at the injection sites.Splitting the components of the vaccine (Id-KLH and adjuvant) in onepatient who had experienced a moderate local reaction and in anotherpatient who had experienced a moderate systemic reaction, characterizedby fever, rigors and diffuse arthralgias, established the adjuvant asthe component associated with these reactions. Both of these moderatereactions resolved completely after 24-48 hours. The only laboratoryabnormality associated with Id immunization was a mild elevation (lessthan twice the normal value) of serum creatine phosphokinase 24 hoursafter immunization in an occasional case.

These results demonstrate that patients with B-cell lymphoma can beinduced to make sustained idiotype-specific immune responses by activeimmunization with purified autologous tumor-derived surface Ig. Theyshow that autologous Id, made immunogenic by conjugation to KLH, canserve as an immunogen (antigen) to elicit host immunological responses.The induction of low levels of idiotype-specific immunity wasdemonstrated in the setting of minimal tumor burden followingconventional chemotherapy. These results, taken together with theinduction of relatively stronger immune responses to the KLH carrier,and exogenous antigen, suggest that chemotherapy-inducedimmunosuppression is not an obstacle to active immunotherapyadministered adjunctively to cytoreductive drug therapy in this manner.

This initial study also established the requirement for an immunologicaladjuvant, as no Id-specific responses were observed prior to theaddition of an adjuvant to the program. The objective of furtherclinical trials using tumor derived Id as a therapeutic vaccine is tofurther optimize the immunogenicity of this vaccine. To this end, thisstudy will focus on the use of novel immunological adjuvants whichare 1) more potent and 2) more effective in the induction ofcell-mediated immune responses, compared with the pluronic polymer-basedadjuvant used in the study.

The 38C13 B cell tumor is used as a model system to screen promisingimmunological adjuvants. A number of these have included cytokines andamong these, GM-CSF has emerged as a promising adjuvant for idiotypic Igantigen. In these experiments (10 mice per group), syngeneic mice wereimmunized with 50 mg Id-KLH derived from the tumor, either alone or incombination with GM-CSF mixed together with the antigen and administeredsubcutaneously. Three additional daily doses of GM-CSF were administereds.c. as close to the original site of immunization as possible. Miceimmunized with an irrelevant Id-KLH (4C5 IgM) served as negativecontrols for the vaccine. Two weeks after this single immunization, allmice were challenged with a single preparation of 38C13 tumor cells(5×10³ cells i.p.) and followed for survival. The results demonstratedthat the augmented survival benefit afforded by immunization withrelevant Id-KLH alone can be significantly enhanced by the addition ofGM-CSF at either the 100 or 10,000 unit dose. The loss of thisprotective effect at a higher dose of GM-CSF of 50,000 units was alsoobserved. These data suggest that GM-CSF may have a potent adjuvanteffect in vivo for Id-KLH antigen, especially at relatively low doses.

The follicular lymphomas are follicular small cleaved cell (FSC) andfollicular mixed lymphoma (FM). Stage I and II patients comprise only10% to 15% of all cases of follicular lymphomas and are best managedwith radiation therapy. Eight-five percent of patients with follicularlymphomas present with stage III or IV disease. The optimal managementof these patients remains controversial and has generally followed twodivergent approaches (16, 17). One is an aggressive approach, which hasincluded radiation therapy, combination chemotherapy, or combinedmodality therapy and the other is a conservative approach that involvesno initial treatment followed by a single-agent chemotherapy orinvolved-field radiotherapy when required (18; 19). Most forms ofsystemic therapy have the capacity to produce high complete responserates. However, they have failed to produce long-term disease-freesurvival or to prolong overall survival; thus, it has become clear thatthe vast majority of patients with this disease will relapse and die oftheir lymphoma, despite its usually indolent course.

The NCI study (MB-110, BRMP 8903) begun in 1978, is a prospectiverandomized study comparing these two distinct approaches to themanagement of stage III or IV indolent histology lymphoma. Most patientswere randomized between no initial therapy or aggressive combinedmodality therapy with ProMACE/MOPP flexitherapy followed by low dose(2400cGy) total nodal irradiation. Among the 149 patients treated thusfar, 125 (84%) were randomized; 62 to watch and wait (W & W) and 63 toaggressive treatment. Among the 62 patients on the watch and wait arm,29 continue to be observed for periods up to 10+ years. The median timeto cross over to aggressive therapy is 23 months.

It is apparent that patients in whom therapy is initiated after thedevelopment of symptoms have a significantly lower complete responserate to therapy than patients randomized to receive the same therapy atdiagnosis (74% vs 40%, P₂=0.0039). The complete responder (CR) rate ofpatients randomized to initial aggressive treatment is comparable tothose obtained in patients with advanced-stage intermediate gradelymphoma receiving the same treatment. The CR rate in indolent lymphomadoes not appear to be significantly higher than what can be achievedwith other combination regimens. For patients randomized to watch andwait, median follow-up of CRs is shorter because of the delay ininitiating treatment. However, the median duration of remission has notbeen reached at five years and 57% of patients are projected to bedisease-free >8 years and 44% are projected to be in a CR at 12 years.The disease-free survival curves are not significantly different betweenthe two arms. Thus, allowing the patient to reach a greater tumor burdenbefore instituting systemic therapy reduces the likelihood of obtaininga CR, but once achieved, CRs are comparably durable to those obtainedfrom primary aggressive therapy. The lengthening of the remissionduration, however, has not resulted in a survival advantage for patientsrandomized to receive primary aggressive chemotherapy. Furthermore, eventhough a minority of complete responders have relapsed, the probabilityof relapse appears to be continuous over time, and the vast majority ofpatients are expected to eventually succumb to their disease.

Thus, even immediate aggressive therapy has not resulted in improvedsurvival. Therefore, although patients diagnosed with follicularlymphoma enjoy relatively longer survival times compared with patientswith solid tumors, follicular lymphoma remains an incurable disease.Novel experimental therapies designed to improve the durability of theremissions already effectively induced by chemotherapy are justified.

Summary of Treatment Plan

The goal is to treat patients with follicular lymphomas to completeremission or maximal response with ProMACE chemotherapy. After thecompletion of chemotherapy, in an effort to reduce the relapse rate (byeradicating microscopic disease resistant to chemotherapy), patientswill receive an autologous Id vaccine administered in combination withGM-CSF.

The goal of this study is to evaluate the ability of the Id vaccine toclear the bone marrow of malignant cells detectable by pathologic(morphologic) examination or molecular examination (polymerase chainreaction, PCR) in patients with PCR amplifiable translocations. Allpatients have serial bone marrow and peripheral blood samples collectedto search for clonal abnormalities by PCR. Patients are followed aftervaccine therapy and their remission status correlated with clinical vs.molecular determinations of response. There should be three categoriesof complete responders: those who had a clinical complete responsebefore the vaccine but had an abnormal clone by PCR that cleared afterthe vaccine; those with a clinical CR before the vaccine who were alsoPCR negative before the vaccine; and those who achieved a clinicalcomplete response but had PCR positive marrows before and after thevaccine. It is a goal of this study to assess whether “molecularcomplete responses” can be achieved using the vaccine in patientsfollowing chemotherapy.

Objectives

The objectives of this trial are to:

To induce cellular and humoral immunity against the unique idiotypeexpressed on the surface of patients' B-cell lymphomas.

To determine the ability of Id immunization to eradicate bcl-2 positivetumor cells from the bone marrow as detected by PCR.

As a secondary objective, to determine the more biologically active ofthe two GM-CSF doses as an adjuvant, as measured by the endpoints in theabove objectives.

To determine the impact of Id immunization on disease free survival ofpatients achieving a CR with chemotherapy.

Patient Selections

Patient Sample

A. Sample size, approximately 42 patients

B. Sex distribution: male and female

C. Age: patients must be ≧18 years old

Eligibility Criteria

-   -   Patient must meet all of the following eligibility criteria:    -   A. Tissue diagnosis of: follicular small cleaved cell, or        follicular mixed lymphoma with surface IgM, IgG or IgA phenotype        with a monoclonal heavy and light chain. Pathology slides must        be submitted to the NIH Pathology Department for review.    -   B. Stage III or IV lymphoma.    -   C. Only previously untreated patients are eligible.    -   D. Previous treatment with radiation alone (less than TBI) is        permissible.    -   E. A single peripheral lymph node of at least 2 cm size        accessible for biopsy/harvest.    -   F. Karnfsky status ≧70%.    -   G. Life expectancy of >one year.    -   H. Serum creatinine ≦1.5 mg/dl unless felt to be secondary to        lymphoma.    -   I. Bilirubin ≦1.5 mg/dl unless felt to be secondary to lymphoma        or Gilbert's disease. SGOT/SGPT <3.5× upper limit of normal.    -   J. Ability to give informed consent. Ability to return to clinic        for adequate follow-up for the period that the protocol        requires.

Patient Exclusion Criteria

-   -   The presence of any exclusion criteria (listed below) will        prohibit entry into study:    -   A. Prior total body irradiation.    -   B. Presence of antibodies to HIV, hepatitis B surface antigen or        other active infectious process.    -   C. Pregnancy or lactation. Fertile men and women must plan to        use effective contraception. A beta-HCG level will be obtained        in women of childbearing potential.    -   D. Patients with previous or concomitant malignancy, regardless        of site, except curatively treated squamous or basal cell        carcinoma of the skin, or effectively treated carcinoma in situ        of the cervix.    -   E. Patient unwilling to give informed consent.    -   F. Failure to meet any of the eligibility criteria described        above.    -   G. Any medical or psychiatric condition that in the opinion of        the protocol chairman would compromise the patient's ability to        tolerate this treatment.    -   H. Patient with CNS lymphoma (current or previously treated)        will not be eligible.

Clinical Evaluation

Complete history and physical examination.

CBC, diff., platelet count.

Serum chemistry, β₂-microglobulin.

PT/PTT

Quantitative immunoglobulins, serum protein electrophoresis,immunoelectrophoresis.

HIV antibody, HBsAg.

Urinalysis.

Serum β-HCG in women of child-bearing potential.

EKG and MUGA.

5 TT for serum storage.

Leukapheresis to obtain 3×10⁹ lymphocytes. These samples will be usedfor baseline studies of T-call activation and response to Id.

Tumor Biopsy—prior to therapy, all patients must undergo biopsy/harvestof a clinically involved peripheral lymph node to obtain tissue formorphological classification, immunophenotypic characterization,determination of immunoglobulin gene rearrangements, bcl-2translocation, cytogenetics, and to provide starting material for an Idvaccine. The sample should be at least 2 cm in size. Only patients withtumors that are surface immunoglobulin positive with a monoclonal heavyand light chain will be accepted as study candidates. Use standardlymphoma vaccine biopsy orders (see protocol below). Leftover tumorbiopsy samples may be used for basic studies of lymphoma biology invitro. Such future studies may be done without re-consenting thesubjects only if the studies involve risks already outlined in theoriginal consent form.

CXR-PA and LAT.

CT scan of abdomen and pelvis.

Lymphangiogram, unless contraindicated by massive pedal edema, severechronic lung disease, ethiodal sensitivity (Note: sensitivity to otheriodine compounds, e.g., renograffin, are relative, but not absolutecontraindications).

Other tests (CT chest, ultrasound, liver scan, bone scan, upper andlower GI series, IVP, MRI) should be performed as needed to evaluate alldisease sites adequately.

Examination of pleural fluid or ascites when present.

Bilateral bone marrow aspirates and biopsies—In addition to the normalaspirate and biopsy, 5 cc of marrow will be aspirated from each sideinto 0.5 ml of PFH for PCR analysis. The procedure should be performedin the usual manner with a biopsy performed first. Then a small volume(0.5-1 cc) can be aspirated for the smear and clot tube. A separateRosenthal needle with bevel should be used for the aspirate. The 5 ccsample for PCR can be obtained from the same site as the initialaspirate.

CT scan of the head and lumbar puncture with CSF analysis if clinicallyindicated.

Patient Registration

Patients will be registered prior to the initiation of therapy at whichtime eligibility criteria will be reviewed. Stratification andrandomization are described in detail below (see Statisticalconsiderations).

Study Design

ProMACE

Day 0 Day 7 Day 28 Cyclophosphamide Cyclophosphamide Next cycle begins650 mg/m² IV 650 mg/m² IV Doxorubicin Doxorubicin  25 mg/m² IV  25 mg/m²IV Etoposide VP-1 6 Etoposide BP-1 6 120 mg/m² IV 120 mg/m² IV

-   -   Prednisone 60 mg/m² po qd×14 (days 0 to 13)    -   Bactrim one double strength tablet po BID throughout therapy

All patients will be treated until a complete remission is obtained andtwo additional cycles of chemotherapy have been given, or until diseasehas been stable for two cycles of chemotherapy, or progressive diseasedevelops. A minimum of six cycles will be given to each completeresponder before therapy is discontinued. Patients with more than 90% PRor a full CR will be continued on the vaccination part of the protocol.Patients with less than 90% PR or progressive disease will be taken offof the study.

Postinduction Therapy—Three to six months (or whenever a customized GMPvaccine is available, up to a maximum period of 12 months) after thecompletion of chemotherapy, all patients in whom either a completeclinical remission or minimal disease status (≧90% partial response) hasbeen achieved will receive a series of five injections of a vaccineconsisting of 0.5 mg autologous tumor derived immunoglobulin (Id)conjugated to KLH. The vaccine will be administered together with GM-CSFas an immunological adjuvant. Both the vaccine and GM-CSF will beadministered subcutaneously according to the following schedule:

Schedule: At 0, 1, 2, 3 and 5 months Id-KLH (0.5 mg s.c.) day 0 adjuvant(s.c.) days 0-3 Cohort 1: GM-CSF 500 mcg/m²/d s.c. for 4 days Cohort 2:GM-CSF 100 mcg/m²/d s.c. for 4 days

The sites of injection will be rotated between the upper and lowerextremities. Each dose of vaccine or GM-CSF will be split equallybetween the two upper or lower extremities. All GM-CSF injections willbe given in close proximity to the vaccination site, as close to theexact site of injection as possible. If local reactions to GM-CSF aresevere, GM-CSF injections may be given elsewhere. Patients will beobserved in the clinic for two hours following Id-KLH and/or GM-CSFadministration. During the observation period, vital signs will be takenevery 15 minutes during the first hour and every 30 minutes during thesecond hour.

Supportive Care

G-CSF 5 mcg/kg/d SC may be used in all patients who are hospitalized forthe treatment of febrile neutropenia, regardless of how long theneutropenia persists.

Grading and Management of Toxicity

Chemotherapy: Dose modification of chemotherapy will be based on thegranulocyte count done at the time of drug administration (day 0 or 7 ofeach cycle). The percentage of drugs administered may be furthermodified based on toxicity in prior cycles (see below). If thegranulocyte count is <1200, and the patient is due for day 0 drugs,delay day 0 for one week until appropriate parameters are met. Ingeneral, delays of up to one week are preferable to starting G-CSF. Ifafter a one week delay, appropriate parameters are still not met, thenG-CSF may be started as above. Also, in general, delays of up to oneweek are preferable to dose reductions. Full doses of all drugs shouldbe given on time if blood count suppression is due to bone marrowinvolvement with disease.

Dose Modification for Hematologic Toxicity

IF GRANULOCYTE THEN DOSE COUNT IS: AS FOLLOWS: On Day 0 ≧1200 100% alldrugs ≦1200 Day 0 Delay

For neutrophil nadir <500 or platelet count <25,000 on previous cycle,75% of cyclophosphamide, doxorubicin, and etoposide should beconsidered. For neutrophil nadir (day 21 counts)>750 on a previouscycle, dose escalation of cyclophosphamide, doxorubicin, and etoposideby 10-20% should be prescribed.

IF PLATELET COUNT IS: THEN DOSE AS FOLLOWS: >100,000 100% of all drugs50-99,999 100% Prednisone  75% Etoposide  50% Cyclophosphamide,Doxorubicin <50,000 Delay

Dose Modification for Non-Hematologic Toxicity

Assessment of non-hematologic toxicity will be graded according to theCRB/DCS/NCI Common Toxicity Criteria. Chemotherapy will be withheld inpatients experiencing grade 2 or greater non-hematologic toxicity untilthe patient has completely recovered from the toxicity. Fornausea/vomiting 2: grade 2, drug therapy should be continued withnon-steroid antiemetics.

Doxorubicin dosage should be adjusted as follows in the presence of thefollowing LFT abnormalities:

% Dose Bilirubin SGOT 100 <1.5 mg/dl <75 U 50 1.5-2.9 mg/dl 75-150 U 253.0-5.9 mg/dl 151-300 U 0 ≧6.0 mg/dl >300 U

Immunotherapy

-   -   Id-KLH Vaccine

Based on previous experience with autologous Id-KLH vaccines, little orno toxicity is expected from the Id-KLH component of the vaccine (15).Nevertheless, any local skin reactions will be carefully noted andscored for erythema, induration, pain and disruption of the barriersurface. If any patient has a reaction suggestive of sensitization, thevaccine may be split into its component parts; specifically, the patientwill be tested with Id-KLH alone and then GM-CSF alone. Toxicities willbe graded according to the CRBINCI/DCS common toxicity criteria.

-   -   GM-CSF

Anticipated toxicities from GM-CSF administration in this dose range areexpected to be mild based on previous experience. Potential toxicitiesinclude fever, chills, myalgias, arthralgias, nausea, vomiting,diarrhea, dyspnea, tachycardia, arrhythmias, elevation of liver functiontests, elevation of BUN and creatinine. However, local skin reactions,such as erythema and induration, may be observed and will be carefullynoted. Attempts will be made to maintain these patients as outpatients.For grade IV fever (not responsive to Indocin or Tylenol), or grade DIvomiting (unresponsive to therapy), GM-CSF will be held until toxicityis less than grade II and will be restarted at 50% of the original doselevel for the rest of that weekly injection cycle and for subsequentcycles. For neurologic toxicity that affects daily function (unable tocarry on simple routine duties, or grade II in the toxicity gradingscale), hold treatment until symptoms resolve, then reduce GM-CSF by50%. If symptoms persist, the adjuvant should be removed for subsequentimmunizations. Patients with grade III neurotoxicity will be removedfrom the study.

For well-documented evidence of cardiac toxicity (i.e., grade III,including evidence of ischemia or ventricular arrhythmia, but notsupraventricular tachycardia or atrial fibrillation controlled bydigoxin or calcium channel blocking agents), the adjuvant will beremoved for subsequent immunizations.

Asymptomatic elevations in serum bilirubin and creatinine (not resultingin hyperkalemia) will be tolerated. For SGOT or SGPT >10× normal, GM-CSFwill be held until values return to <5× normal, then resumed at 50% ofthe GM-CSF dose for all remaining doses.

Fever and chills associated with vaccine administration and/or GM-CSFwill be treated with TYLENOL and/or DEMEROL. The use of non-steroidalantiinflammatory drugs and/or steroids should be avoided. Shouldnon-steroidals or steroids be required for unrelated medical conditionsfor a course exceeding 2 weeks, the patient will be taken off of thestudy.

Adverse Drug Reactions

All toxicities and adverse events will be recorded on the study flowsheet and appropriately graded as to severity and cause. Toxicities thatare related to the underlying disease should be clearly differentiatedfrom drug toxicities.

Adverse drug reactions related to chemotherapy will be submitted basedon guidelines for commercial drugs.

Reports of adverse reactions to Id-KLH and GM-CSF will be made using theDivision of Cancer Treatment Common Toxicity Criteria for referenceaccording to the guidelines published by the DCT, NCI. These guidelinescan be summarized as follows:

-   -   A. Report by telephone to IDB within 24 hours (301) 230-2330        -   1. All life-threatening events (grade 4, except for grade 4            myelosuppression) which may be due to administration of the            investigational drug(s),        -   2. All fatal events (grade 5),        -   3. All first occurrences of any previously unknown toxicity            (regardless of grade).    -   B. A written report should follow within 10 working days.    -   C. All adverse drug reactions will also be reported in writing        to the NCI Institutional Review Board within 10 working days.    -   D. All adverse drug reactions will also be reported to the FDA        in accordance with Federal regulations.    -   E. Data will be submitted at least every two weeks.

Study Parameters

During Chemotherapy

-   -   Weekly: CBC, diff. platelets; except day 14, i.e. CBC on day 0,        7, 21, and 28.    -   Beginning of each cycle: Chem 20, CXR, LAG follow-up (KUB), CT        scans (only after 4 cycles, then every 2 cycles).    -   Bilateral bone marrow aspirate and biopsy after four cycles and        every additional two cycles thereafter. Include 5 cc of aspirate        in PFH from each side for PCR analysis.

At Maximal Response to Chemotherapy

-   -   If residual disease is obvious, record measurements and perform        bone marrows as above.    -   For complete responders, complete restaging should be performed.        This should include all studies that were positive at initial        staging evaluation with the exception of repeat thoracotomy or        laparotomy. Bilateral bone marrows should be performed as above.

During Vaccine Therapy

-   -   If residual disease is obvious, record measurements and perform        bone marrows as above.    -   PT-PTT day 0    -   UA, β₂ microglobulin day 0 of each immunization.    -   Leukapheresis is performed on the day of initiation of vaccine        therapy (prior to the first cycle only) to obtain pre-vaccine        lymphocytes for storage. Five tiger top tubes are drawn at this        time to obtain serum for storage.    -   Two tiger top tubes and peripheral blood (60 cc in PFH) are        collected on day 0 of each monthly cycle, for preparation of        serum and lymphocytes, respectively.    -   Skin Biopsy is obtained near a planned immunization site on day        0 prior to the first cycle (baseline sample) and again on day 1,        2, or 3 of cycle 3 at an active site of erythema and/or        induration as close to the original biopsy site as possible.    -   DTH—Delayed type hypersensitivity test (DTH) to autologous        idiotype protein is performed during cycle 4 and again following        completion of the immunization regimen, i.e., during or after        cycle 5. The DTH-test is performed by intradermal injection of        0.5 mg of idiotype protein in 0.1-0.2 ml of NS. To ascertain the        specificity of a positive reaction, 0.5 mg of a heterologous        isotype matched Id-protein (from another patient on the same        study) in the same volume will be used as a negative control.        The control idiotypes used on these two occasions will be from        two different patients, also in the study, in order to minimize        the possibility of eliciting an immunologic response against a        particular irrelevant idiotype. A skin biopsy will also be        obtained at the site of the intradermal injection of idiotype        protein and at the control site, one to three days, after the        intradermal injections.    -   Fine needle aspiration or core biopsy (with or without CT        guidance) of any enlarged lymph node draining the vaccination        sites is performed to obtain lymphocytes for in vitro assays.

At Discontinuation of Vaccine

-   -   Restaging as described for Chemotherapy (see “At Maximal        Response to Chemotherapy,” above).    -   Bilateral bone marrow aspirates and biopsies at completion of        therapy and every six months for two years after completing        therapy and yearly thereafter.    -   10 cc of serum for storage and 60 cc of peripheral blood in PHI        is collected at completion of therapy and every three months for        a year.

Specimen Processing and Immunological Assays

Lymph Node Harvest/Biopsy

Each lymph node biopsy will be divided as follows: (a) one-third of thespecimen will be sent in saline to the Hematopathology Section,Laboratory of Pathology, NIH. Biopsies are processed for routinehistopathy and for immunophenotypic characterization, particularly withrespect to monotypic heavy and light chain expression; and (b)two-thirds of the specimen is sent in sterile saline in a sterilecontainer to Clinical Immunology Services, NCIFCRDC, where it isprocessed into a single-cell suspension and cryopreserved.

Blood and Bone Marrow Samples

All peripheral blood and bone marrow aspirate samples are sent in anexpedited manner to Clinical Immunology Services, NCI-FCRDC. Tiger toptubes are spun down and serum divided into 1 ml aliquots for frozenstorage. Peripheral blood mononuclear cells (PBMC) are isolated prior tofreezing by Ficoll-hypaque centrifugation using standard protocols.

Assay for Serum Antibody

In a direct enzyme-linked immunosorbent assay (ELISA), preimmune andhyperimmune serum samples from each patient are diluted over wells of amicroliter plate that are coated with either autologous immunoglobulinidiotype or a panel of isotype-matched human tumor immunoglobulins ofunrelated idiotype. Bound antibody is detected with horseradishperoxidase-goat antihuman light-chain antibodies directed against thelight chain not present in the immunoglobulin idiotype (CaltagLaboratories, South San Francisco).

Assay for Idiotype-Specific Proliferative Response

Whenever feasible, fresh PBMC, isolated above, are used on the same daythey are obtained. Stored frozen PBMC are available as a back-up. PBMCare washed and plated at a concentration of 4×10⁵ cells per well inIscove's modified Dulbecco's medium (IMDM) with 1 percent human AB7serum (IMDM-1 percent AB). KLH, autologous immunoglobulin idiotype, or apanel of isotype matched immunoglobulins of irrelevant idiotypes atconcentrations of 0 to 100 μg per milliliter in IMDM-1 percent ABpreparation are added in triplicate. After the cells are incubated forthree days at 37° C. in an atmosphere containing 5 percent carbondioxide, they are transferred to a preparation of IMDM and 5 percentfetal-.calf serum containing recombinant interleukin-2 (30 Uppermilliliter). The plates are incubated for two days and pulsed for 16 to20 hours with ³H-labeled thymidine (1 μCi per well). Data are expressedas mean (±SEM) counts per minute of [³H]thymidine incorporation.

Initial five-day cultures of PBMCs established as described above areexpanded in IMDM-5 percent fetal-calf serum containing interleukin-2 (30Upper milliliter). Harvested cells are replaced in IMDM-1 percent ABcontaining autologous immunoglobulin idiotype and fresh irradiated (5000R) autologous PBMCs (4×10⁵ cells per well) as antigen-presenting cellsfor five days, before pulsing with ³[H]thymidine.

Cytotoxicity Assays

The potential cytotoxicity of PBMC cultured with Id as above, or withirradiated fresh cryopreserved tumor cells, is assayed against eitherautologous lymphoblastoid cell lines (LBL) pulsed with Id or freshcryopreserved tumor targets. Autologous LBL pulsed with soluble antigenhave been used successfully as targets to detect gp 160-specificcytotoxic T-lymphocytes (20). Historically, the inability to establishlong-term cultures of follicular lymphoma has hindered theiravailability as targets. However, two recent reports have described theuse of fresh cryopreserved lymphoma cell targets, with levels ofspontaneous incorporated radioisotope release in the acceptable range of<35% (21-22). Standard four hour ⁵¹Cr release, as well as 18-24 hour¹¹¹In release assays are used.

Autologous LBL are prepared from pre-immune PBMC by the AIDS MonitoringLaboratory, NCI-FDRDC, using published methods.

Monitoring of T-cell Receptor (TCR) Status

Pre-chemotherapy and pre- and postimmunization serum samples are assayedfor TCR status by Western blot assay. Approximately 7×10⁶ purifiedT-cells from PBMC are lysed for 5 minutes at 4° C. in lysis buffer (25mM Tris, pH 7.4 [Sigma Chemical Co., St Louis, Mo.], 300 mM NaCl, 0.05%Triton X-100, 1 mM Na orthovanadate, 10 μg/ml aprotinin, 10 μg/mlleupeptin, 10 mM nitrophenol-guanidine benzoate [NPGB] and 5 mM EDTA).The lysates are centrifuged at 12,000 rpm at 4° C. for 5 minutes andsupernatant is removed with a micropipettor, making sure the nuclearpellet is not disturbed. A sample of the supernatant is then used toquantitate protein using the BCA protein assay (Pierce, Rockford, Ill.).The rest of the lysate is boiled with 3× reducing sample buffer for 5minutes and placed on ice before its use in Western blot.

Varying concentrations of cellular lysate ranging between 1 and 30 μgare electrophoresed in 14% Tris-glycine gels (Novex ExperimentalTechnology, CA) under reducing conditions and then transferred toImobilon-p PVDF transfer membranes (Millipore Co., Bedford, Mass.). Themembranes are incubated with a 5% solution of non-fat dried milk for onehour and then blotted for one hour at room temperature with anti-TCRCanti-serum (Onco-Zeta 1, OncoTherapeutics, Cranbury, N. J.) at a 1:2000dilution. The membranes are washed with TBS-T buffer [1 M Tris base, 5MNaCl, 0.1% Tween 20 (pH 7.5)] and incubated with anti-rabbit oranti-mouse Ig horseradish peroxidase (Amersham, Buckinghamshire, UK).After washing with TBS-T, the membranes are developed with thechemiluminescence kit ECL (Amersham, UK) for 1-5 minutes. X-OMAT AR film(Kodak Co., Rochester, N. Y.) is used to detect the chemiluminescence.

PCR Amplification of Rearranged bcl-2

Nested oligonucleotide amplification is performed at the MBR or mcr ofthe bcl-2/Ig_(H) hybrid gene using previously published methods (23).Briefly, samples containing 1 μg of genomic DNA are initially amplifiedfor 25 cycles in a final volume of 50 μg containing 50 mmol/L KCl, 10mmol/L Tris HCL, 2.25 mmol/L MgCl₂, 200 mmol/L oligonucleotide primers,200 mmol/L each of dGTP, dCTP, dTTP and dATP, and 1.5 U Taq polymerase(Cetus, Emeryville, Calif.). Reamplification of an aliquot of product isperformed for 30 cycles in a final volume of 50 μl using identicalconditions to the original amplification, with oligonucleotide primersinternal to the original primers. Aliquots of the final product areanalyzed by gel electrophoresis in 4% agarose gels containing ethidiumbromide and visualized under UV light. DNA is Southern blotted ontoZeta-probe blotting membrane (BioRad. Richmond, Calif.) andbcl-2-specific DNA is detected by hybridization with oligonucleotideprobes radiolabeled with ³²P(ATP) using T4 polynucleotide kinase.

Removal of Patients from Protocol Therapy

Patients will be removed from protocol for any of the following reasons:

Unacceptable toxicity (as defined above).

The patient declines further therapy.

The patient experiences progressive lymphoma.

It is deemed in the best interest of the patient. In this instance,

-   -   The Principal Investigator should be notified.    -   The reasons for withdrawal should be noted in the flow sheet.

Response Criteria

Patients will be reevaluated for tumor response after every two cyclesof chemotherapy using the following criteria:

Complete Response—disappearance of all clinical and laboratory(excluding PCR) signs and symptoms of active disease for a minimum ofone month.

Partial Response—a 50% or greater reduction in the size of the lesionsas defined by the sum of the products of the longest perpendiculardiameters of all measured lesions lasting for a minimum of one month. Nolesions may increase in size and no new lesions may appear.

Minimal Residual Response—a ≧90% partial response. For most patients inthis category, this will mean ≦10% residual bone marrow involvement bylymphoma.

Progressive Disease—an increase of 25% or more in the sum of theproducts of the longest perpendicular diameters of all measuredindicator lesions compared to the smallest previous measurement or theappearance of a new lesion.

Drug Formulation and Toxicity Data

Cyclophosphamide (CTX. Cytoxan)—NSC #26271

-   -   Source and Pharmacology—CTX is an alkylating agent, related to        nitrogen mustard, which is biochemically inert until it is        metabolized to its active components by the liver        phosphoramidases. It is non-phase-specific. The drug is excreted        exclusively by the kidney after parenteral administration.    -   Formulation and Stability—CTX is supplied as a 100, 200, 500,        1000 mg and a 2 gram lyophilized powder with 75 mg mannitol per        100 mg (anhydrous) cyclophosphamide. The vials are stored at        room temperature (59-86° F.) and reconstituted with sterile        water for injection to yield a final concentration of 20 mg/ml        as described in the package insert. Reconstituted        cyclophosphamide is stable for at least 6 days under        refrigeration and for 24 hours at room temperature.        Reconstituted drug and diluted solutions should be stored under        refrigeration.    -   Supplier—Commercially available.    -   Route of Administration—The cyclophosphamide used in this        regimen is given IV over 30 minutes and is diluted in 100 cc of        either D₅W or NSS.

Toxicity—Toxicities described with cyclophosphamide include nausea,vomiting, myelosuppression, gonadal failure in both males and females,alopecia, interstitial pneumonitis, pulmonary fibrosis. hemorrhagiccystitis, cardiac events (cardiomyopathy), syndrome of inappropriateantidiuretic hormone secretion (SIADH) and rarely, anaphylaxis.

Prednisone (Deltasone, Meticorten, Liquid Pred) NSC#10023

-   -   Source and Pharmacology—Prednisone is the synthetic congener of        hydrocortisone, the natural adrenal hormone. It binds with        steroid receptors on the nuclear membrane, blocks mitosis, and        inhibits protein synthesis. It kills primarily during the        S-phase of the cell cycle. It is catabolized in the liver and        excreted in the urine. Peak blood levels occur within two hours        after oral intake. Plasma half-life is 3-6 hours. (Biologic        half-life is 12-30 hours.)

Cortisone 25 Equivalent Hydrocortisone 20 strength in mg Prednisone 5Decadron 0.75

-   -   Formulation and Stability—Available in 1, 2.5, 5, 10, 20 and 50        mg tablets; 5 mg/5 ml liquid.    -   Supplier—Prednisone is commercially available.    -   Route of Administration—PO; NOTE: May cause GI upset; take with        meals or snacks. Take in the morning prior to 9 a.m.    -   Toxicity—Toxicities described with prednisone include fluid and        electrolyte changes, edema, hypertension, hyperglycemia,        gastritis, osteoporosis, myopathy, behavioral and mood changes,        poor wound healing, and Cushing's syndrome (moon face, buffalo        hump, central obesity, acne, hirsutism and striae).

VP-16 (Etoposide.VePesid) NSC#141540

-   -   Source and Pharmacology—VP-16 is a semisynthetic derivative of        podophyllotoxin which inhibits topoisomerase II and functions as        mitotic inhibitor, but does not bind microtubules. Its main        effect appears to be in the S and G₂-phase of the cell cycle.        The mean terminal half-life is 11.5 hours, with a range of 3 to        15 hours. It is primarily excreted in the urine.    -   Formulation and Stability—VP-16 is supplied in vials containing        either 100 or 500 mg of etoposide (20 mg/ml) in a polyethylene        vehicle. VP-16 is diluted in either 500 cc of 5% dextrose or        0.9% Sodium Chloride Injection. Diluted solutions        (concentrations of 0.2, 0.4 mg/ml and 1 mg/ml) are stable for        96, 48 hours and 2 hours, respectively at room temperature under        normal room fluorescent light in both glass and plastic        containers. Do not refrigerate etoposide-containing solutions.    -   Supplier—VP-16 is commercially available.    -   Route of Administration—Etoposide is administered as an IV        infusion over 60 minutes.    -   Toxicity—Toxicities described with etoposide administration        include myelosuppression (neutropenia), nausea, vomiting,        mucositis, allergic reactions characterized by anaphylactic        symptoms and hypotension and alopecia.

Doxorubricin (Adriamycin) NSC #123127

-   -   Source and Pharmacology—Doxorubicin is an anthracycline        antibiotic isolated from cultures of Streptomyces peucetius. It        binds to DNA and inhibits nucleic acid synthesis, with its major        lethal effect occurring during the S-phase of the cell cycle:        Since it is primarily excreted by the liver, any liver        impairment may enhance toxicity. Some of the drug has a very        short α T ½ of <20 minutes and a β½ of 17 hours. Animal studies        indicate cytotoxic levels persist in tissue for as long as 24        hours. Biliary excretion also is a source of elimination for        Doxorubicin; therefore, patients with        hyperbilirubinemia/cholestasis caused by something other than        lymphoma should have dosage modification.    -   Formulation and stability—Doxorubicin is available as a        freeze-dried powder in 10, 50 and 150 mg vials. The drug is        stored at room temperature, protected from light, and is        reconstituted with sodium chloride 0.9% (NSS) to yield a final        concentration of 5 mg/ml. The reconstituted solution is stable        for 7 days at room temperature (15-30° C.) or if stored under        refrigeration (2-8° C.).    -   Supplier—Doxorubicin is commercially available.    -   Route of Administration—Doxorubicin is given as a slow IV        injection over 5-7 minutes through an established line with a        free flowing IV.    -   Special precautions: Avoid extravasation and local contact with        skin or conjunctiva.    -   Toxicity—Toxicities described with doxorubicin administration        include myelosuppression, nausea, vomiting, mucositis,        stomatitis, alopecia, diarrhea, facial flushing, dose-related        congestive cardiomopathy, arrhythmias, vein streaking        (hypersensitivity reaction), radiation-recall dermatitis, local        cellulitis, vesication and tissue necrosis upon extravasation        (SQ and dermal necrosis).

ID-KLH Vaccine

-   -   Source—Idiotype protein from the individual B cell lymphomas is        obtained from tissue culture, purified, and covalently coupled        to keyhole limpet hemocyanin (KLH) as previously described. Each        batch is produced according to Good Manufacturing Practices        standards and tested for sterility, endotoxin contamination, and        general safety prior to its use in any patient. The preparation        and quality control/quality assurance testing of the Id-KLH        conjugate is performed by TSI Washington under CRB contract. The        IND for the Id-KLH vaccine will be held by the Drug Regulatory        Affairs Section, CTEP.    -   How supplied—Formulated product for subcutaneous administration        contains 0.5 mg of Id and KLH each per ml of normal saline.        Id-KLH is supplied as a 1 ml vial.    -   Storage—Prior to administration, Id-KLH is stored at −20° C.    -   Administration—After thawing and gentle agitation, the vial        contents are drawn up using an 18-gauge needle on a syringe.        After the entire contents have been drawn up, the 18-gauge        needle is replaced by a 25-gauge needle for injection. This        procedure is important to ensure that all particulates (normal        components of this vaccine) are obtained from the vial.    -   Toxicity—Toxicities described with Id-KLH vaccine administration        include local site reactions (erythema, induration, swelling and        tenderness), fever, chills, rash, myalgias and arthralgias. Mild        elevations in creatinine phosphokinase (CPK) have been observed.

GM-CSF (Sargramostim: NSC #613795; BB-IND 2632

-   -   Source and Pharmacology—The GM-CSF used in this study is        glycosylated, recombinant human GM-CSF. This GM-CSF is an        altered form of the native molecule; the position 23 arginine        has been replaced with a leucine to facilitate expression of the        protein in yeast (Saccharomyces cerevisiae).    -   Formulation and Stability—The GM-CSF is formulated as a white        lyophilized cake and is provided in vials containing 500 μg of        the GM-CSF protein as well as 10.0 mg of sucrose, 40.0 mg of        mannitol, and 1.2 mg of Tris (Trimethamine).    -   To prepare a vial of GM-CSF for direct subcutaneous use,        aseptically inject 1.0 ml of Sterile Water for Injection, USP,        into the vial to dissolve the lyophilized cake. The diluent        should be directed against the side of the vial to avoid excess        foaming. Avoid vigorous agitation of the vial; do not shake.        This yields a solution containing 500 μg/ml. The unreconstituted        material should be kept refrigerated at 2-8° C. and is stable        for at least eighteen months. Once reconstituted, the solution        is stable for at least 24 hours at 2-8° C. or at 18-25° C.        Because the product does not contain a preservative, vials        should be treated as unit-dose containers; reconstituted        solution should be held at 2-8° C. and discarded after no more        than six hours. Do not freeze GM-CSF.

Supplier. Manufactured by Immunex.

-   -   Route of Administration—The appropriate total dose is withdrawn        into and administered from a plastic tuberculin syringe. The        GM-CSF is injected subcutaneously as close as possible to the        Id-KLH injection site. All GM-CSF doses for each patient are        administered by the nursing staff in the outpatient unit.    -   Toxicity—Toxicities described in patients receiving GM-CSF        include: fever, chills, diaphoresis, myalgias, fatigue, malaise,        headache, dizziness, dyspnea, bronchospasm, pleural effusion,        anorexia, indigestion, nausea, vomiting, diarrhea, injection        site tenderness, urticaria, rash, pruritus, hypersensitivity        reaction, bone pain, thromboembolic events, phlebitis,        hypotension, peripheral edema, leukocytosis, thrombocytosis or        thrombocytopenia, hepatic enzyme abnormalities, and bilirubin        elevation. The first administration of GM-CSF has provoked a        syndrome of dyspnea and hypotension within two hours after        GM-CSF injection in a single patient receiving yeast-derived        GM-CSF; this type of reaction has more frequently been observed        in patients receiving GM-CSF produced in E. coli. One report of        a vascular leak-like syndrome occurring after autologous bone        marrow transplant in a patient receiving continuous IV infusion        of GM-CSF has been recorded.

Unconjugated Lymphoma Immunoglobulin Idiotype (for intradermal skintesting) NSC#684151

-   -   Source—The patient-specific purified idiotype protein,        previously produced according to GMP standards as described        above, is vialed as a separate product by TSI Washington        Laboratories and will be supplied by CTEP, DCT, NCI. This vialed        product is tested separately for sterility, endotoxin, and        mycoplasma, according to IND specifications previously discussed        with the FDA.    -   Each vial of patient-specific unconjugated idiotype will be        labeled to include the following information:        -   Purified sterile immunoglobulin idiotype patient-specific            lot        -   final volume and concentration of product        -   patient-specific immunoglobulin subtype        -   storage conditions        -   fill date        -   patient identification (first name/last initial)    -   How Supplied—This product is available as a solution containing        0.2-0.3 ml of unconjugated idiotype diluted in sodium chloride        0.9%. The solution is contained inside a sterile vial. The final        solution contains 0.5 mg of patient-specific immunoglobulin        idiotype protein. Intact vials are stored at −20° C.    -   Toxicity—The toxicities associated with administration of        unconjugated Id protein are anticipated to be identical to those        described with the Id-KLH vaccine.        -   The safety issues regarding the injection of heterologous            idiotype protein isolated from other patients' B-cell tumors            have already been fully addressed in CRB #9407 (NCI            T94-0085; Active immunization of Healthy Sibling Marrow            Transplant Donors With Myeloma-derived Idiotype) and are            felt to be minimal, because of the highly purified nature of            the protein.        -   Briefly, an immune response of any consequence to the            isotype matched idiotype used as a negative control during            the second skin test is not likely, based on:        -   1. The isotype matched idiotype will only be administered            once and is not conjugated to a carrier protein. These            minimize the chance of eliciting a sustained immune response            to the protein.        -   2. Any immune response specifically directed against the            idiotype (i.e., variable region) on the control idiotype            protein is not likely to cross-react with host cells and is            therefore not likely to be of any consequence.        -   3. An autoimmune response against constant region or            allotype determinants shared between the idiotype of the            patient's own tumor and that of the control idiotype tumor            is theoretically possible. However no evidence of such            autoimmune responses have been observed either in vivo or in            vitro during the course of immunization of sibling bone            marrow transplant donors with purified myeloma protein.        -   Furthermore, a safety precedent exists for immunizing            patients with material derived from tumor cells from other            patients. For example, in attempting to develop immune            responses against metastatic melanomas, patients were            immunized with 1) intact melanoma cells; 2) shed antigens            fractionated by detergent treatment and            ultracentrifugation; 3) melanoma cells infected with            vaccinia virus and melanoma cells freeze thawed and            mechanically disrupted, all using a pool of allogeneic            melanoma cell lines (24-28).

Bactrim will be supplied by the Clinical Center.

Filgrastim (G-CSF)/Neupogen

-   -   Source and Pharmacology—The G-CSF to be used in this study is        the recombinant methionyl human granulocyte-colony stimulating        factor (r-methi-HuG-CSF). G-CSF is a hematopoietic growth factor        with effects on both immature bone marrow progenitors and mature        myeloid cells. It acts by supporting growth of human bone marrow        derived colony forming units and enhancing neutrophil growth and        proliferation.    -   Formulation and Stability—The G-CSF is formulated as a clear,        sterile solution and is provided in vials at a final        concentration of 300 mcg/ml. The commercial vials are available        in 300 and 480 mcg sizes. The intact vials are stored under        refrigeration (2-8° C.) prior to use and must not be frozen and        are stable at this temperature for at least one year.    -   Supplier—Manufactured by Amgen; supplied by the Clinical Center.    -   Route of Administration—The appropriate total dose is withdrawn        into and administered from a plastic tuberculin syringe. The        G-CSF is injected as a subcutaneous injection. The patient or        other care-giver is instructed on proper injection technique.    -   Toxicities—Toxicities described with G-CSF include: transient        bone pain (sternal/pelvic) myalgias, fatigue, mild elevations in        uric acid, LDH and alkaline phosphate, fluid retention,        transient hypotension, local inflammation at injection site,        rarely cutaneous vasculitis, rarely pericardial effusion and        rare anaphylactic reactions with first dose.

Statistical Considerations

Statistical issues to be addressed include identification of significantendpoints, sample size determination, power considerations,stratification, randomization and design.

The design of this study is viewed primarily within the framework of aSingle Arm Phase II trial. However, as the purpose is also toinvestigate possible differences between GM-CSF doses as adjuvants, itincorporates design elements characteristic of a Multiple Arm Phase IIor a randomized Phase Di trial. Statistical methods that are appropriateto both single and double arm designs are described.

Patients receive combination chemotherapy to best response followed byId-KLH combined with GM-CSF. Several outcome measures (endpoints) areevaluated. in order to meet the objectives of this study. They include:

1) The clinical complete response rate (in contradistinction to themolecular or PCR response rate) of all patients to ProMACE—a percentageindicated by the disappearance of all clinical and laboratory signs andsymptoms of active disease, excluding PCR, for a minimum of one month.

2) The Polymerase Chain Reaction (PCR) response rate (molecular−completeresponse rate)−the percentage of patients who, having achieved aclinical complete response still remain PCR (+) at the end ofchemotherapy, and who then become PCR (−) with the administration ofimmunotherapy.

3) Disease Free Survival Rate—computed by Kaplan-Meier curves andrelated survival measures.

The PCR response rate is taken as the primary outcome variable ofinterest to ascertain the following: (1) to determine the ability of Idimmunization to eradicate bcl-2 positive tumor cells from the bonemarrow and; (2) to identify the more biologically active of the twodoses of GM-CSF. In this endeavor, the plan is to accrue 42 patients. Itis estimated that approximately 38 (90%) of these patients will be bcl-2(+) and thus evaluable for molecular response rate. The other fourpatients may still be evaluable for a molecular response rate based onIg gene amplification using allele-specific (CDR3) primers by PCR. Fromprevious experience with ProMACE-based regimens, it is estimated that 32(85%) of these patients will achieve either a complete response(complete clinical response, CCR) or a partial response in which a >90%partial remission has been obtained (high partial response, HPR). Theaccuracy of these estimates are of some interest. For the 42 (90%)patients anticipated to be bcl-2 (+), lower and upper 95% confidenceintervals are 77% and 96%. For the 38 (85%) patients anticipated toachieve either a complete clinical response or a high partial response,the lower and upper confidence intervals are 70% and 93%.

Patients are stratified on the basis of their ProMACE treatmentperformance as either a complete clinical responder (CCR) or as a highpartial responder (HPR). It is not known exactly what percentage ofthese 32 patients will be CCRs and what percentage will be HPR'S. Hencea block size of four (4) is used in the randomization scheme to assure areasonably balanced allocation to each dose group. Given the patientsallocation stratum, he (she) is randomly assigned to one of the adjuvantgroups according to the envelope method (29). Specifically, a block offour assignments is placed in four separate envelopes. The block of fouris placed in one of the two allocation strata, say CCR. Another block offour is placed in the other allocation strata, say CCR. Another block offour is placed in the other allocation stratum, HPR. When a patient isto be randomized, a call is made to the biostatistician who, after beinginformed of the patients status as either a CCR or an HPR, randomlydraws an envelope from the appropriate stratum to determine the patientsdose group assignment. After the four envelopes pertinent to aparticular stratum have been exhausted, the next batch of four envelopesis made available for use. This procedure is continued until a total of32 patients have been assigned to the two dose groups.

For example, it is estimated that 50-80 percent of pathological completeresponders will fall into the CCR category. If 75% of 32, or 24 patientswere to be classified as CCRs, six blocks of four envelopes would berequired to randomly assign 12 patients to cohort 1 and 12 patients tocohort 2. A similar procedure would occur concurrently with the 8patients classified as HPRs. Two blocks of four envelopes would berequired to randomly assign 4 patients to cohort 1 and 4 patients tocohort 2. At no time could the number of patients in each dose groupdiffer by more than four.

At the time of data analysis, approximately 16 subjects will compriseeach dose group and a test for the difference in PCR response ratesbetween the two groups will be conducted. By hypothesis, neither dosegroup is predicted to have a higher PCR response rate than the other;hence, a two-tailed test is appropriate. Power calculations show that,with the groups limited to 16 patients, the difference in PCR responserates will have to be large (30, 31). For example, to detect adifference at the α=0.05 level of significance with power (1-β) equal to80%, the response rates must differ by 55%; with power equal to 50%, theresponse rates must differ by 50%. In the event that no significantdifference is detected, the subjects will be pooled and the overall PCRresponse rate will be assessed. With a total of 32 CCRs and HPRs treatedwith vaccine, the width of a two-tailed 95% confidence interval for aresponse rate of 50% will not exceed 17 percentage points. If the actualresponse rate is higher or lower than 50%, the confidence interval willbe smaller.

Disease-free survival distributions are estimated by the Kaplan-Meier(product-limit) method and dose groups are compared using the log ranktest. If no dose group differences are detected, the subjects from bothgroups are pooled and the Kaplan-Meier estimate of the survivorshipfunction and related functions are evaluated. If suggested by the dataanalysis, parametric distributions (e.g., Weibull, log-normal) are fitas well (32, 33).

Research ethics: Subjects from [both genders and] all racial/ethnicgroups are eligible for this study if they meet the eligibility criteriaoutlined above. To date, there is not information that suggests thatdifferences in grud metabolism or disease response would be expected inone group compared to another. Efforts are made to extend accrual to arepresentative population, but in this preliminary study, a balance mustbe struck between patient safety considerations and limitations on thenumber of individuals exposed to potentially toxic and/or ineffectivetreatments on the one hand and the need to explore gender and ethnicaspects of clinical research on the other hand. If differences inoutcome that correlate to gender or to ethnic identity are noted,accrual can be expanded or a follow-up study can be written toinvestigate those differences more fully. Alternatively, substantialscientific data exist demonstrating that there is no significantdifference in outcome between genders or various ethnic groups.

Records to be Kept and Quality Assurance

Consent form: The original signed informed consent documents will bekept with the patient's other study documentation (e.g., the researchchart). A copy of the informed consent document will also be retained bythe Data Management Section.

The Clinical Coordinator, Data Management Section, will ascertain thedates of the IRB approvals before registering the first patient.

It is understood that the disclosed invention is not limited to theparticular methodology, protocols, and reagents described as these mayvary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “ahost cell” includes a plurality of such host cells, reference to “theantibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are as described. Publications cited herein andthe material for which they are cited are specifically incorporated byreference. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

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1. A fusion polypeptide comprising a viral chemokine and a human tumorantigen, wherein the viral chemokine comprises MC148.
 2. The fusionpolypeptide of claim 1, wherein the human tumor antigen comprises ahuman B cell lymphoma tumor antigen.
 3. The fusion polypeptide of claim1, wherein the human tumor antigen comprises human Muc-1.
 4. The fusionpolypeptide of claim 1, wherein the human tumor antigen comprises theamino acid sequence of SEQ ID NO:
 1. 5. An isolated nucleic acidmolecule encoding the fusion polypeptide of claim
 1. 6. A vectorcomprising the nucleic acid molecule of claim
 5. 7. An isolated hostcell comprising the vector of claim
 6. 8. A composition comprising thefusion polypeptide of claim 1 in a pharmaceutically acceptable carrier.9. A method of producing an immune response in a subject, comprisingadministering to the subject the composition of claim
 8. 10. The methodof claim 9, wherein the immune response is an effector T cell immuneresponse.
 11. The method of claim 9, wherein the subject has a tumor.12. The method of claim 11, wherein the tumor is a B cell tumor.
 13. Themethod of claim 12, wherein the human tumor antigen is a B cell tumorantigen.
 14. The method of claim 13, wherein the B cell tumor antigen isselected from the group consisting of: (1) an antibody produced by a Bcell tumor; (2) a single chain antibody comprising linked VH and VLdomains which retain the conformation and specific binding activity ofthe native idiotype of the antibody produced by a B cell tumor; and (3)an epitope of an idiotype of an antibody produced by a B cell tumor. 15.A composition comprising the nucleic acid molecule of claim 5 in apharmaceutically acceptable carrier.
 16. A method of producing an immuneresponse in a subject, comprising administering to the subject thecomposition of claim 15, under conditions whereby the nucleic acid ofthe composition can be expressed.
 17. The method of claim 16, whereinthe immune response is an effector T cell immune response.
 18. Themethod of claim 16, wherein the subject has a tumor.
 19. The method ofclaim 18, wherein the tumor is a B cell tumor.
 20. The method of claim19, wherein the human tumor antigen is a B cell tumor antigen.
 21. Themethod of claim 20, wherein the B cell tumor antigen is selected fromthe group consisting of: (1) an antibody produced by a B cell tumor; (2)a single chain antibody comprising linked VH and VL domains which retainthe conformation and specific binding activity of the native idiotype ofthe antibody produced by a B cell tumor; and (3) an epitope of anidiotype of an antibody produced by a B cell tumor.